65
ISBN: 92–9146–760–x
Manual for addressing major 
INITIATIVE ON
Sustainable Animal
Productivity reproductive problems and 
mastitis in dairy animals 
CGIAR’s Sustainable Animal Productivity for Livelihoods, 
Nutrition and Gender inclusion (SAPLING) is working 
in seven countries focusing on livestock value chains to in Nepal
package and scale out tried-and-tested, as well as new, 
innovations in livestock health, genetics, feed and market systems. SAPLING aims to demonstrate that improvements in 
livestock productivity can offer a triple win: generating improved livelihoods and nutritional outcomes; contributing to 
women’s empowerment; and, reducing impacts on climate and the environment. Its seven focus countries are Ethiopia, 
Kenya, Mali, Nepal, Tanzania, Uganda and Vietnam. 
I L R I   M A N U A L
Manual for addressing major reproductive 
problems and mastitis in dairy animals in Nepal 
Ram Pratim Deka, Naba Kumar Das, Laxman Prasad Ghimire and Mamta Pathak
International Livestock research Institute
December 2022
INITIATIVE ON
Sustainable Animal 
Productivity
©2022 International Livestock Research Institute (ILRI) 
ILRI thanks all donors and organizations which globally support its work through their contributions to the CGIAR Trust Fund
This publication is copyrighted by the International Livestock Research Institute (ILRI). It is licensed for use under   
the Creative Commons Attribution 4.0 International Licence. To view this licence, visit https://creativecommons.
org/licenses/by/4.0. 
Unless otherwise noted, you are free to share (copy and redistribute the material in any medium or format), adapt (remix, 
transform, and build upon the material) for any purpose, even commercially, under the following conditions:
 
ATTRIBUTION. The work must be attributed, but not in any way that suggests endorsement by ILRI or the author(s).
NOTICE:
For any reuse or distribution, the licence terms of this work must be made clear to others. 
Any of the above conditions can be waived if permission is obtained from the copyright holder. 
Nothing in this licence impairs or restricts the author’s moral rights. 
Fair dealing and other rights are in no way affected by the above. 
The parts used must not misrepresent the meaning of the publication.  
ILRI would appreciate being sent a copy of any materials in which text, photos etc. have been used.
Editing, design and layout—ILRI Editorial and Publishing Services, Addis Ababa, Ethiopia.
Cover photo: ILRI/ Susan MacMillan.
ISBN: 92–9146–760–x
Citation: Deka, R.P., Das, N.K., Ghimire, L.P. and Pathak, M. 2022. Manual for addressing major reproductive problems and mastitis in 
dairy animals in Nepal. ILRI Manual 65. Nairobi, Kenya: International Livestock Research Institute (ILRI).
Patron: Professor Peter C Doherty AC, FAA, FRS 
Animal scientist, Nobel Prize Laureate for Physiology or Medicine–1996
Box 30709, Nairobi 00100 ilri.org Box 5689, Addis Ababa, Ethiopia 
Kenya better lives through livestock Phone +251 11 617 2000 
Phone  +254 20 422 3000  Fax +251 11 667 6923 
Fax      +254 20 422 3001 ILRI is a CGIAR research centre Email ilri-ethiopia@cgiar.org 
Email ilri-kenya@cgiar.org
ILRI has offices in East Africa • South Asia • Southeast and East Asia • Southern Africa • West Africa
Manual for addressing major reproductive problems and mastitis in dairy animals in Nepal iii
Contents
Tables iv
Figures v
Abbreviations vi
Acknowledgements vii
1 Background 1
2 Reproductive problems/disorders 4
2.1. Anoestrus 4
2.2. Repeat breeding (RB) 12
2.3. Abortion 18
2.4. Stillbirth 23
2.5. Retained placenta 24
2.6. Purulent vaginal discharge 26
2.7. Dystocia 27
2.8. Mastitis 30
3. Process to be followed for per rectal examination 36
3.1. Materials required for rectal examining a cow/buffalo cow 36
3.2. Personal precaution during rectal examination 37
3.3. Procedure for rectal examination 37
4. Sample collecting, labelling, storing and dispatching 38
4.1. Steps in collecting samples to dispatching to laboratory: 38
4.2. Procedure for collecting vaginal/uterine discharge: 38
4.3. Procedure for collecting milk sample: 39
4.4. Procedure for collecting sample for PCR: 42
4.5. General considerations in sample collecting, handling and storage: 43
4.6. Sample labelling and storing: 43
4.7. Packaging and dispatching samples to other laboratories: 44
Annex I. Animal observation check list 45
References 48
iv Manual for addressing major reproductive problems and mastitis in dairy animals in Nepal
Tables
Table 1. Observing various indicators of reproductive performance 6
Table 2. Differential diagnosis from pregnancy 10
Table 3. Differential diagnosis between follicular cyst, luteal cyst and cystic corpus luteum 11
Table 4. Differential diagnosis from repeat breeding  15
Table 5. Differential diagnosis of causes of abortion 21
Manual for addressing major reproductive problems and mastitis in dairy animals in Nepal v
Figures
Figure 1. Good growth of calves in accordance with age. 5
Figure 2. A well-nourished heifer with proper growth. 5
Figure 3. Handling the reproductive organs a large animal  4. 
Figure 4. Various stages of follicular development during oestrus  5
Figure 5. Follicular cyst. 7
Figure 6. Luteal cyst. 8
Figure 7. A cystic corpus luteum (CCL). 8
Figure 8. A Doppler ultrasonography machine. 9
Figure 9. Persistent corpus luteum. 9
Figure 10. An aborted foetus. 18
Figure 11. A case of retained placenta. 24
Figure 12.Developmental defect: Hydrocephalic foetus removed by foetotomy 28
Figure 13. Developmental defect: Conjoined twin 28
Figure 14. Dicephalic monster. 28
Figure 15.  Abnormal foetal presentation: Breech presentation 28
Figure 16. Abnormal Abnormal foetal posture: Bilateral shoulder flexion 28
Figure 17. Mastitis. 31
Figure 18. A CMT kit.  34
Figure 19. Interpreting results from CMT. 34
Figure 20. Per rectal exploring a cow/buffalo cow.  36
Figure 21. Materials required for rectal examining a cow/buffalo cow. 36
Figure 22. Clean udder. 40
Figure 23. Cleaning of the teat. 41
Figure 24. Collecting milk sample in a sterile milk vial. 41
Figure 25. Milk sample. 41
vi Manual for addressing major reproductive problems and mastitis in dairy animals in Nepal
Abbreviations
AI Artificial Insemination
CCL Cystic Corpus Luteum
CL Corpus luteum
CMT California Mastitis Test
COD Cystic Ovarian Degeneration
CS Caesarean Section
DCP Digestible Crude Protein
EDTA Ethylenediaminetetraacetic acid
ELISA Enzyme-linked immunosorbent assay 
GnRH Gonadotrophin Releasing Hormone
HCG Human Chorionic Gonadotrophin
LH Lutenizing Hormone
MRT Milk Ring Test
MoALD Ministry of Agriculture and Livestock Development
NDDB National Dairy Development Board
PCL Persistent Corpus Luteum
PCR Polymerase Chain Reaction
PG F2α Prostaglandin F2α
RB Repeat breeding
RBPT Rose Bengal Plate Test
ROP Retention of Placenta
SCC Somatic Cell Count
SCM Subclinical mastitis
TDN Total Digestible Nutrient
 
 
 
Manual for addressing major reproductive problems and mastitis in dairy animals in Nepal vii
Acknowledgements
This research was conducted under the CGIAR Initiative on Sustainable Animal Productivity. CGIAR research is 
supported by contributions from the CGIAR Trust Fund. CGIAR is a global research partnership for a food-secure 
future dedicated to transforming food, land, and water systems in a climate crisis.
Manual for addressing major reproductive problems and mastitis in dairy animals in Nepal 1
1 Background
A cross sectional study was conducted from May 2016 to January 2017 in Rubavu and Nyabihu districts, Western 
Rwanda, aiming at estimating the prevalence of subclinical mastitis (SCM) and identifying its causative bacteria. 
Managing practices and milking procedures were recorded through a questionnaire. One hundred twenty-three 
crossbreed milking cows from 13 dairy farms were randomly selected and screened for SCM using California Mastitis 
Test (CMT). Composite CMT positive milk samples were processed for bacterial isolation and identification. 
The overall SCM prevalence at cow level was 50.4%. Sixty-eight bacterial isolates were identified by morphological 
and biochemical characteristics. They included, Coagulase Negative Staphylococci (51.5%), Staphylococcus 
aureus (20.6%), Streptococcus species (10.3%), Bacillus species (10.3%), Streptococcus agalactiae (5.8%) 
and Escherichia coli (1.5%). About 67.1% of the farmers checked for mastitis; of these, 58.9% relied on clinical signs 
and only 6.8% screened with CMT. Only 5.5% and 2.7% of the farmers tried to control mastitis using dry cow therapy 
and teat dips, respectively. Thus, to reduce the prevalence of SCM, farmers in the study area need to be trained on 
good milking practices, including regular use of teat dips, applying dry cow therapy and SCM screening. This will 
improve their sales and their financial status.
Dairying plays an important role in the livelihood and nutrition of Nepali people. In Nepal, dairy sector contributes 
about 9% to total GDP (NDDB, 2021) wherein buffalo production contributes 57.23% of the total milk and  and 
36.13% of the total meat (MoALD, 2020)   As such, buffalo is considered as one of the most important livestock 
species in the country.
In Nepal, a considerable number of households are involved in dairy farming. Many of them consider buffalo 
farming as a main source of livelihood. In Nepal, 65% of the buffaloes are indigenous and rest 35% are cross breeds 
(Neopane, 2006). Based on their breed characteristics, buffaloes are broadly divided into three groups viz. Hill 
buffalo, Terai buffalo and Indian breeds (Rasali, 1997). Among the Hill buffaloes, Lime, Parkote and Gaddi are 
considered to the important breeds.  Lime buffaloes are generally characterized by grey colour with chevron marks 
around the brisket and hugely found in northern hills whereas, Parkote buffaloes are predominantly dark in colour 
with no chevron marks and localized more towards the southern midhills (Rasali et al. 1998; Amatya et al. 2000). 
Gaddi buffaloes are found in the far Western Development Region (Pokhrel et al. 1998).
Terai buffaloes are largely considered as nondescript type. Apart from indigenous buffaloes, about 10% in the hills 
and little over 10% in the Terai are said to be of Indian Murrah breed or their crosses which have come to exist in the 
various pocket areas of the country as a result of crossbreeding program and occasional imports of buffaloes from 
India (Lamsal et al. 2020). 
The productivity of buffaloes is lower than that of dairy cows. It is mainly due to lower reproductive efficiency. 
Delayed sexual maturity, postpartum anoestrus, silent oestrus and the seasonal breeding pattern are some of the 
reasons behind reduced reproductive efficiency of buffaloes. Among these, infertility, manifested by anoestrus, 
remains as a major problem in buffaloes. There is paucity of reports in Nepal pertaining to economic impact of 
anoestrus in buffaloes. 
In India, Kumar 2013 reported an estimated loss of 372.90 Indian rupees (INR) per day from each buffalo due to 
anoestrus (USD 1.00 = INR 81.6694 at 16 January 2023). As the incidence of anoestrus has been reported high, the 
above figure shows great economic impact at the country level. Considering the total buffalo population in Nepal, 
2 Manual for addressing major reproductive problems and mastitis in dairy animals in Nepal
its milk production should have much higher than the existing one. This could not have achieved mainly because of 
higher incidences of various reproductive diseases/disorders and higher prevalence of mastitis.
Reproductive health of a dairy buffalo is a condition in which buffalo cow is physically sound enough to meet the 
normal parameters of reproduction such as attaining puberty in time, conceiving at the first attempt of natural service 
or artificial insemination (AI) (in the second heat), maintain full term of pregnancy with optimal weight gain of the 
calf, parturition without difficulty, shedding of placenta on time, coming into next heat within the specific time, 
maintaining normal inter calving period etc. Any deviation from this may be due to reproductive diseases/disorders 
such as anoestrus, repeat breeding (RB), abortion, retained placenta, vaginal discharge, dystocia etc. These invite 
serious attention of the owners as well as of the veterinarians. If not addressed properly in time, the reproductive 
disease/disorders will cause poor productive and reproductive performances, resulting in higher economic loss.
Mastitis is the most widespread and major disease of dairy cattle, causing huge loss to the dairy farmers. Mastitis 
not only reduces milk yield but also has a negative effect on milk composition. Because of mastitis, the cost of milk 
production and spoilage increases. Moreover, mastitis has a serious threat of zoonosis associated with shedding of 
bacteria and their toxins in the milk. Mastitis requires proper management at both clinical and subclinical stages for 
its control and prevention.
To bring these two harmful elements under control for augmenting milk production, a treatment and management 
protocol is the urgent need of the hour so that these two issues can be addressed uniformly across the nation.
Frequently encountered reproductive problems in dairy animals in Nepal
A fairly good number of reproductive problems/disorders of the female reproductive organs are commonly 
observed in dairy animals which are responsible for significant loss to the dairy farmers. These reproductive 
problems/disorders include:
a. Anoestrus 
• Silent heat
• Delayed onset of puberty
• Delayed postpartum oestrus
b. Repeat breeding (RB)
• Delayed ovulation 
• Anovulatory heat
• Endometritis 
c. Prolapse (vaginal or uterine)
d. Abortion
e. Stillbirth
f. Retained placenta
g. Vaginal discharge
h. Dystocia
Manual for addressing major reproductive problems and mastitis in dairy animals in Nepal 3
Some important terminologies used in the manual
In this protocol, some important terminologies have been used quite commonly which needs some clarity at this 
stage. The terminologies have been stated below:
• Mastitis: It is inflaming the mammary gland (udder and teat).
• Uterine involution: Restoring the uterus to its normal nonpregnant size and function after parturition is termed 
as uterine involution. Involution process depends on the rate of myometrial contractions, eliminating bacterial 
infection and the histological regenerating the endometrium.
• Puerperium: Puerperium is the postnatal period beginning from birth of a calf up to six weeks of age.
• Metritis: It is inflaming the uterus.
• Endometritis: It is inflaming the endometrium.
• Cervicitis: It is inflaming the cervix.
• Abortion: Expulsion or loss of a dead or live recognizable size of foetus at any stage of gestation from the age of 
45 days up to parturition from the uterus before full term of gestation is called abortion.
• Early embryonic death: Pregnancy lost before 42 days of pregnancy is considered as early embryonic death.
• Stillbirth: Expulsion of dead foetus between 260 days and parturition is called stillbirth.
• Mycotoxins: Toxins produced by certain species of fungus.
• Metabolic diseases: Metabolic disease refers to a group of conditions caused by certain essential nutrients 
resulting in disturbance of the animals’ normal metabolic processes.
• Pyometra: Forming and accumulating pus in the uterus. This inhibits secreting Prostaglandin F2 α (PG F2α), which 
block regression of the corpus luteum (CL) and prevent oestrus. There are generally no accompanying clinical 
signs other than anoestrus.
• Mucometra: It is accumulating thin or viscid fluid in the uterus. It results from cystic ovaries leading to cessation of 
heat.
• Corpus luteum (CL): A corpus luteum is a mass of cells that forms in an ovary and is responsible for producing the 
hormone progesterone during early pregnancy.
4 Manual for addressing major reproductive problems and mastitis in dairy animals in Nepal
2 Reproductive problems/disorders
2.1. Anoestrus
Anoestrus may be defined as the failure of the heifer/buffalo cow to come to oestrus/heat within the normal range of 
time. It is of two types: 
• delayed onset of puberty (in heifers) and 
• delayed postpartum oestrus.
2.1.1. Delayed onset of puberty
• A cattle/buffalo heifer maintained under a proper nutritional plan, ideally attains puberty within 21–24 months of 
age which may go up to 36 months in case of indigenous heifers. When the period is further extended, it may be 
considered as delayed onset of puberty.
2.1.1.1. Possible causes of delayed onset of puberty
• Poor nutrition/undernutrition during the growth period
• Parasitic infestation
• Anatomical defects of the genital tract
• Improper ovarian function
• Hormonal imbalance
• Persistent hymen/white heifer disease
• Genetic effects
2.1.1.2. Approach to disease investigation
Step I: Taking animal history (in the record sheet given at Annex I)
• Age of the animal
• Breed of the animal
• System of rearing (intensive/semi-intensive)
• Deworming done in last one year
• Feeds and feeding regime (concentrate, green and dry fodder with quantity andf requency)
• Supplementing nutrition (i.e. minerals, vitamins, probiotics etc.)
• Providing exercise if any
Manual for addressing major reproductive problems and mastitis in dairy animals in Nepal 5
• Proximity of bull with the heifer
• Experiencing detecting heat symptom by the farmer.
Step II: Clinical observation external observation
• Colour of visible mucous membranes—pale mucous membranes indicate anaemia.
• Body coat (rough or smooth)—rough body coat indicates malnutrition, parasitic infestation etc.
• Growth in accordance with age (Figure 1).
Figure 1. Good growth of calves in accordance with age.
Figure 2. A well-nourished heifer with proper growth.
Figure 3  Handling the reproductive organs  Figure 4. Various stages of follicular development 
of a large animal during oestrus.
  
Per rectal examination
As showed in Figures 3 and 4, observing reproductive organs like ovaries, development of follicles, uterine horns 
etc. (Table 1).
6 Manual for addressing major reproductive problems and mastitis in dairy animals in Nepal
Table 1. Observing various indicators of reproductive performance 
Organs to be observed Observation Interpretation
Colour of visible mucous Pale Anaemia
membranes
Body coat Rough Malnutrition, parasitic infestation etc.
Shiny, smooth Well nourished
Ovaries Smaller in size and with a smooth surface Non-functional ovaries or absence of ovary
Uneven surface Indicative of follicular development or CL
Palpable growth over the ovarian surface -do-
Uterus Tonic uterus Physiologically active uterus
Vulvar lips Well developed Nearing oestrus
2.1.1.3. Possible line of treatment
Step I: If nutritional deficiency is observed
• Should give dewormer at periodic interval.
• May provide minerals, vitamins like A, D3, E preparations (both oral and parenteral) as per the need.
• Should provide balanced concentrate feed.
Step II: If poor follicular growth is observed
• May give utero-ovarian massage for seven alternative days followed by applying Lugol’s paint into the os 
cervix.
Step III: If hormonal cause is anticipated
• May give hormonal treatment, if necessary.
• Review after 30 days, if any development of reproductive organs is observed, a single dose of PG F2α can be 
administered.
2.1.1.4. Advice to the farmers
• Maintain proper plan of nutrition. Concentrate feed should be given to the heifers along with addition of 50 gm of 
mineral mixture daily.
• May increase concentrate ration from the 18 months of age in case of heifer.
• Give deworming drugs periodically.
• Allow for regular exercise.
• Keep the heifer close to bull, it may hasten puberty.
• May cull the animal if it does not respond to treatment.
Manual for addressing major reproductive problems and mastitis in dairy animals in Nepal 7
2.1.2. Delayed postpartum oestrus
Normally a buffalo cow comes to heat within 28–71 days after parturition, which may be extended up to 90 days. 
If it takes even more longer time, it is considered as delayed postpartum oestrus. It is of two types—suboestrus 
(unobserved or silent heat) and true anoestrus
Suboestrus: It is also known as silent heat. In this case, buffalo cows do not exhibit external behavioural symptoms 
of oestrus or heat, although the physiological symptoms of oestrus are present internally. Because of this, farmers fail 
to go for AI or natural service. This could be detected only if a bull is present in the vicinity.
True anoestrus: In this case, buffalo cow fails to come to heat because of physiological, hormonal, or organic 
changes that are required to bring the animals into heat. Internally, ovary is found subfunctional and corpus luteum 
and follicle are not palpable.
2.1.2.1. Possible causes of delayed postpartum oestrus
Improper involution of the uterus: Involution of the uterus is necessary for the buffalo cow to conceive again at 
postpartum. Time required for complete uterine involution in a normal cow generally ranges from 26 to 52 days 
which may be extended up to 90 days. Sometimes, because of some factors, it goes far beyond. These factors 
include:
• Weak myometrial contraction
• Incomplete removal of the placental tissues/membrane
• Slower regeneration of the epithelium of the endometrium
• Seasonal influence (involution is rapid in spring and summer)
• Nutritional status
• Uterine infection
• Metabolic diseases (milk fever increases the uterine involution period).
Cystic ovarian degeneration (COD): On clinical examining a buffalo cow showing cyclic irregularity characterized 
by anoestrus or nymphomania may have one or more fluid-filled ovarian structures (cysts). These cysts may be of two 
types:
Follicular cyst—causes nymphomania and repeat breeding (Figure 5).
Figure 5. Follicular cyst.
8 Manual for addressing major reproductive problems and mastitis in dairy animals in Nepal
Luteal cyst—It causes anoestrus. Luteal cysts may disappear spontaneously because of the release of endogenous 
PG (Figure 6).
Figure 6. Luteal cyst.
Cystic corpus luteum (CCL)—A CL (corpus luteum) with a cavity or lacunae having fluid arises after normal ovulation 
(Figure 7). It makes actual size of CL will be small that’s why progesterone hormone will be less which causes 
embryonic death and anoestrus or repeat breeding.
Figure 7. A cystic corpus luteum (CCL).
Pregnancy diagnosis
Pregnancy diagnosis is a difficult task at the initial stage of pregnancy. It becomes even more difficult, when buffalo 
cow gets pregnant by bull service without the notice of the owner. Under such circumstances, farmers may 
wrongly consider the case as a delay of postpartum oestrus which may invite unnecessary concern/care/treatment. 
Therefore, proper pregnancy diagnosis is essential at the initial stage. Following pregnancy diagnostic methods 
could be employed for this purpose.
Rectogenital palpation—It is the oldest method of pregnancy diagnosis and is the easiest, cheapest and fastest 
method of pregnancy diagnosis with little or no harm to the dairy animal and its foetus when performed carefully.
Ubio Quickvet bovine pregnancy spot assay—It is a qualitative enzyme immunoassay for detecting progesterone 
in milk in 15–20 minutes within 21 days of pregnancy. It measures progesterone levels in milk or serum (for heifers) 
using a rapid Enzyme-linked immunosorbent assay (ELISA) technique.
Limitation: The test should be conducted between 19th and 23rd days after insemination.
Doppler ultrasonography machine—It is a portable ultrasonography machine. Using it, pregnancy can be 
detected in a cow/buffalo cow starting from the 28th day with 100% accuracy (Figure 8). 
Manual for addressing major reproductive problems and mastitis in dairy animals in Nepal 9
Figure 8. A Doppler ultrasonography machine.
Limitation: The cost is high.
Persistent Corpus luteum (PCL): When Corpus luteum fails to regress spontaneously within the specified time, 
there is continuous secretion of progesterone which inhibits postpartum oestrus (Figure 9).
Figure 9. Persistent corpus luteum.
Buffalo cow–calf association: Late weaning causes delay in postpartum oestrus and vice versa. If suckling 
is continuously practiced during lactation, it inhibits ovulation, elongates period of postpartum anoestrus and 
contributes to delayed postpartum oestrus.
Subnutrition/undernutrition—A poor plan of nutrition with mineral deficiency delays postpartum oestrus.
Parasitic infestation—Parasitic infestation in buffalo cause anaemia and weight loss and ultimately results into 
anoestrus.
Pyometra—Inhibits secretion of PG F2α which in turn blocks regression of the CL to cause anoestrus. There are 
generally no accompanying clinical signs other than anoestrus.
Mucometra—It results from cystic ovaries leading to cessation of heat.
2.1.2.2. Approach to disease investigation 
Step I: Taking animal history
• Date of last calving
• Whether primipara or pluripara
• History of retained placenta if any
10 Manual for addressing major reproductive problems and mastitis in dairy animals in Nepal
• Nutritional plan offered
• History of metabolic diseases if any
• Date of weaning
• Date of deworming
Step II: Observing clinical findings
External observation:
• Vaginal/uterine discharge
Per rectal examination
• For presence of ovarian cyst or PCL
• For presence of pus/mucus in the uterus
• Pregnancy diagnosis
Step III: Laboratory investigation
Possible tests to differentiate among follicular cyst, luteal cyst and cystic corpus luteum.
• ELISA test is performed for estimating progesterone concentrations by an enzyme immunoassay before and at 10 
days after cow/buffalo cow is treated with Gonadotrophin releasing hormone (GnRH). 
Sample required: Blood serum sample.
Differential diagnosis
To make differential diagnosis between pregnancy and pyometra, the following comparisons could be made (Table 
2).
Table 2. Differential diagnosis from pregnancy
Criteria Pregnancy Pyometra
Uterine horns Horns remain asymmetrical Both the uterine horns remain equally 
distended
Fremitus Fremitus is present Fremitus is absent because there is no 
need to supply extra blood
Slippage of foetal membrane Slippage of foetal membrane is present No slippage of foetal membrane
Consistency of uterine wall Thin and tonic uterine wall Thick uterine wall and lack of tone
Differential diagnosis of follicular cyst, luteal cyst and cystic corpus luteum
To make differential diagnosis among follicular cyst, luteal cyst and cystic corpus luteum, milk progesterone 
concentrations should be estimated by an enzyme immunoassay before and at 10 days after buffalo cows are treated 
with gonadotropin-releasing hormone (Table 3).
Manual for addressing major reproductive problems and mastitis in dairy animals in Nepal 11
Table 3. Differential diagnosis between follicular cyst, luteal cyst and cystic corpus luteum 
Criteria Follicular cyst Luteal cyst Cystic corpus luteum
Before 10 days After 10 days Before 10 days After 10 days Before 10 days After days 10
External symptoms Nymphomania Anoestrus Anoestrus
Progesterone in  Less than 1.0 1.0 ng/ml or Increased  1.0 ng/ml or Decreased value
concentration skim milk ng/ml higher value higher
Consistency on rectal Soft, thin walled Firm, thick walled Very soft, thin 
palpation Farin et al. structure structure walled in the 
(1992) top of CL
2.1.2.3. Possible line of treatment 
Step I:
In case of PCL
• May perform manual enucleation of CL
• May inject PG F2α intramuscularly (i/m)
• May give single i/m injection of GnRH analogue (10 to 20 μg).
In case of pyometra/mucometra
• Conservative treatment such as uterine irrigation could be done.
• Oestrogen/PG F2α could be injected followed by uterine irrigation.
• Intrauterine antibiotic and antiprotozoal drug combination like Ciprofloxacin andT inidazole, Ofloxacin and 
Ornidazole, Povidone iodine (50:50) etc. could be used.
• Parenteral antibiotics could be used if any infection is suspected.
• Some b u f f a l o  cows m a y  fail to evacuate pus c o m p l e t e l y  after the first treatment. In suchc ase, 2nd 
injection of PG F2α may be repeated 8 to 12 days after the first dose.
In case of COD
• Manual enucleation: Manual enucleation of cystic structures could be done by palpation perr ectum. However, it 
may cause ovarian haemorrhages and adhesions may follow manual rupture, so it should be avoided.
• GnRH analogue: Receptal (Buserelin) could be injected and cow/buffalo cow may show sign of oestrus in 
between 18 to 23 day of post treatment.
• Mineral supplementation could be given.
2.1.2.4. Advice to the farmers
• Go for early weaning.
• Call a veterinarian/paraveterinarian at time of/after calving so that proper postpartum care can be taken.
• Complete the course of treatment of metabolic disease, if any, as advised byt he attending veterinarian.
• Make deworming plan with appropriate medicine and proper dose. 
12 Manual for addressing major reproductive problems and mastitis in dairy animals in Nepal
• Perform faecal test before and after deworming to find out the efficacy of medicine.
• Maintain a proper plan of nutrition along with mineral supplementation.
• Call a veterinarian for palpation to find out the condition before culling.
• Cull the animals if it does not respond to treatment within six months.
2.2. Repeat breeding (RB)
RB may be defined as a buffalo cow’s failure to conceive from three or more regularly spaced natural services/AI 
without exhibiting any detectable abnormalities. 
2.2.1. Possible causes of RB
Subnutrition/undernutrition during the growth period
• Undernourished cows/buffalo cows show poor rates of conception. Buffalo cows not receiving balanced ration 
may become repeat breeder.
• Trace minerals particularly copper, cobalt, iron etc. are essential for steroidogenesis, i.e. formation of sex 
hormones, deficiency of which may lead to RB.
Delayed ovulation
• In majority of buffalo cows, ovulation normally takes place approximately 24–48 hours after the onset of oestrus or 
preovulatory Lutenizing Hormone (LH) surge, i.e. 15–18 hours after external signs of oestrus have ceased. 
• In some buffalo cows, ovulation normally takes place beyond 6–21 hours (average 18 hours) after external signs 
of oestrus have ceased. In such case, the spermatozoa fail to fertilize the ova as it loses its viability beyond the 
specific time.
Anovulatory heat
• The ovary fails to release ovum at time of heat.
Early embryonic death within 16 days after service
• Stress such as pain, long transportation etc.
• Malnutrition
• Seasonal stress such as summer stress
• Progesterone deficiency
• Uterine infection.
Age
The older the buffalo cow, the higher is the chances of RB. Fertility in dairy buffalo cows get better after the 1st or 2nd 
parturition and then declines from the 6th and 7th parturition.
Manual for addressing major reproductive problems and mastitis in dairy animals in Nepal 13
Uterine infection
• There should be a favourable uterine environment for normal embryonic development. Any adverse condition 
such as cervicitis, endometritis, pyometra, metritis, trichomoniasis, vibriosis (now, campylobacterosis) etc. 
adversely affects the normal uterine condition causing embryonic death which is one of the major causes of RB.
• Uterine infections negatively influence the postpartum uterine involution and follicular development.
Anatomical defects of the genital tract
• Blocked fallopian tubes due to infection or physiological defect.
Improper ovarian function
• COD is a cause of RB in dairy animals.
• Delayed ovulation.
• Luteal inadequacy resulting into progesterone deficiency.
AI
• Poor quality of semen may reduce the conceiving rate.
• Thawing in appropriate temperature 35°C for 35 seconds. 
• Depositing semen in body of cervix or last ring of cervix for higher conception.
• After completing AI, watch inside of the sheath, so that all semen is deposited or not.
• If animals are not inseminated at accurate time (12–18 hours from onset of heat), it may cause conception failure.
• Poor management and handling of frozen semen.
• Storing frozen semen in insufficient level of liquid nitrogen.
• Breaking cold chain.
• Contaminated AI instruments.
Prolonged oestrus period
• In some dairy animals, the oestrus period continues for more than the normal 24 hours, even up to 72 hours. 
In such case, AI timing should be scheduled as required and number of AI shall be more than once or twice. If 
buffalo is in heat after 6 hours of service, repeat the service after 10 to 12 hours from the 1st service.
Service by an infected breeding bull
• A bull infected with sexually transmissible disease like—Trichomoniasis, Brucellosis, Vibriosis etc. will transmit the 
disease to the cow/buffalo cow during service and results in RB of the cow/buffalo cow.
2.2.2. Approach to disease investigation 
Step I: Taking animal history
• Age of the cow/buffalo cow.
• General health condition of the cow/buffalo cow (poor/good/very good).
14 Manual for addressing major reproductive problems and mastitis in dairy animals in Nepal
• Any chronic disease being suffered by the animal (e.g. diarrhoea, cough, fever etc.).
• Normal appetite of the animal.
• Nutritional plan (concentrate, green and dry fodder, minerals and vitamins supplemente tc.) offered.
• Number of times the animal has repeated.
• Sign of heat observed.
• Date and time of each services given to the animal (to see whetherr epeating in regular or irregular oestrus).
• Difference between the time of sign of heat observed and insemination/serviceg iven.
Step II: Clinical observation of the animal 
External:
• Examining vulvo vaginal area for presence of any abnormal discharge.
• Examining vulvular mucosa by opening the vulva after three days of breeding to find out 1st degree of metritis 
(pus flakes is observed).
• Per rectal examination for presence of abnormal discharge from vagina, cervix andu terus (cloudy, bloody, watery, 
mixed with pus etc.).
Per rectal examination:
• Per rectal examination for presence of abnormal discharge (in minute quantity) in the cervix/vaginal canal/uterus 
using AI gun and AI sheath.
Procedure of examination:
• Get the uterine catheter properly sterilized.
• Open the vulvar lips and insert the uterine catheter into the cervix of the cow/buffalo cow and push gently up to 
the 1st one-third.
• Holding in the grasp of the uterus with the left hand, gently make short to and fromm ovement of the catheter 
with the right hand.
• Gently withdraw the uterine catheter and observe for presence of any discharge—cloudy, bloody, watery, mixed 
with pus etc.
Or, in case a uterine catheter is not readily available
• Insert an AI gun into the cervix of the cow/buffalo cow and push gently up to the 1st one-third.
• Withdraw the AI gun carefully leaving the sheath inside the cervix.
• Grasp the uterus with the left hand and gently move the sheath with the right hand.
• Gently withdraw the sheath and observe the presence of any discharge— cloudy,b loody, watery, mixed with 
pus etc.
• If it is not transparent, it may be assumed to have some kind infection in ther eproductive tract.
Or observe the discharge by back racking
• Per rectal examination of reproductive organs like— ovaries for presence of cyst etc.
Manual for addressing major reproductive problems and mastitis in dairy animals in Nepal 15
• Per rectal examination of ovaries on 9th or 10th day of oestrus for presence of corpus luteum. If corpus luteum is 
not present on the ovary, it is a case of anovulatory heat.
Step III: Laboratory investigation
Possible tests
• Culture and sensitivity test
• ELISA
• Polymerase Chain Reaction (PCR)
Sample required
• Culture and sensitivity test— Aseptically collected vaginal/uterine discharge.
• ELISA— Blood serum sample.
• PCR— Blood samples collected using tubes with Ethylenediaminetetraacetic acid (EDTA).
• Transporting to the hospital in cool temperature using cool box/thermosflask.
• Packaging and dispatching to laboratory following standard protocol as early asp ossible in thermocol box 
adding ice pack/gel pack.
Differential diagnosis (Table 4)
Table 4. Differential diagnosis from repeat breeding 
Conditions Observations Interpretation
Interval between two Normal cycle (18–22 days) Anovulatory heat, delayed ovulation, faulty AI, 
consecutive heat progesterone deficiency
Beyond 22 days Uterine infection such as cervicitis, endometritis, 
pyometra, metritis etc.
35–55 days Trichomoniasis
Less than 18 days Follicular cyst
Type of vaginal Transparent and watery Early heat
discharge Transparent and viscous, cuts off in the midway Late heat
Profuse watery Mucometra
Cloudy and with variable degree of consistency Infection in the vagina, cervix or uterus
White or whitish-yellow, mucopurulent Severe endometritis
Frequent and copious Follicular cyst
Bloody Acute and severe endometritis
External symptoms of No bellowing, not restless Silent heat
heat Tries to mount on other cow/buffalo cow, Normal heat
profound signs like bellowing,running, 
restlessness, vaginal discharge
At frequent intervals, very restless Follicular cyst, buller cow
Appearance of the  Smooth, small Anovulatory heat
ovary on palpation Presence of soft, thin walled cyst Follicular cyst
Duration of oestrus More than 24 hours Prolonged oestrus period
16 Manual for addressing major reproductive problems and mastitis in dairy animals in Nepal
2.2.3. Possible line of treatment 
Step I: Curative treatment
In case of undernutrition
• Prescribe a proper plan of nutrition.
In case of delayed ovulation
• Inseminating the cow/buffalo cow two or three times at 12 hours interval.
• Injecting Human Chorionic Gonadotrophin (HCG) 4 hours before AI. 
• Providing vitamins A, D3, E and minerals like phosphorus, selenium, iron, copper, cobalt etc. parenterally and 
orally one week prior to breeding.
In case of anovulatory heat
• GnRH analogues or HCG (2000 LU i/m or 1000 LU i/v) at the time of insemination to promote ovulation.
• Provide vitamins A, D3, E and minerals like— phosphorus, selenium, iron, copper, cobalt etc. parenterally and 
orally one week prior to breeding.
In case of early embryonic death due to external or environmental factors
• Proper management may help to overcome this defect.
In case of early embryonic death due to heat stress
• Inseminated cow/buffalo cow should be kept in a cool place or in a shed for 15 days after insemination.
• Administering GnRH analogue (2.5 ml) on day 11 after insemination.
• Providing adequated rinking water and may cool the shed with water.
In case of progesterone deficiency
• Administer progesterone injection 3 to 5 days after insemination and continued for av ariable period of 2 to 3 
weeks.
In case of mild/subclinical uterine infection (oestrus cycle is generally regular)
• Intrauterine deposition of Levofloxacin, Ornidazole and Alpha Tocopherolc ombination, Ciprofloxacin and 
Tinidazole combination, 5–6 hours prior to AI.
• Parenteral use of Enrofloxacin, Ciprofloxacin, Ceftriaxone etc. as per body weight fort hree days.
• Inject 500 mg progesterone i/m after 5 days of insemination, repeat the same doseo n day 10.
In case of severe uterine infection (oestrus cycle is irregular/prolonged)
• May irrigate the uterus with potassium permanganate solution or isotonic saline solution; if required, clean the 
uterus of any pus, debris, caseated/necrotic materials etc.
• May inject Oxytocin i/v to induce uterine contractions for complete evacuation of uterus.
• May deposit intrauterine—Levofloxacin, Ornidazole and Alpha Tocopherol combination, Ciprofloxacin and 
Tinidazole combination.
Manual for addressing major reproductive problems and mastitis in dairy animals in Nepal 17
• May irrigate uterus with povidone iodine 1–2% solution in 1st degree, 2–3% solution. In 2nd degree 3–4% 
solution for 3rd degree of metritis @ 50–100 ml daily for 3 days. Povidone iodine is highly effective in the 
presence of pus, debris and blood in the uterus while other antimicrobials lose their effectiveness. Povidone 
iodine always uses in luteal phase for better response.
• May administer Enrofloxacin, Ciprofloxacin, Ceftriaxone etc. as per body weight for three days.
• May give a course of anti-inflammatory drug in case of severe inflammation and pain.
• May inject vitamins A, D3 and E weekly for three weeks or alternate days.
• Should allow sexual rest in the subsequent heat.
• Should allow service on the 3rd heat, followed by injecting 1500 IU HCG after AI.
• May inject 500 mg progesterone i/m after 5 days of insemination, may repeat the same cow/buffalo cow dose on 
day 10.
In case of severe/acute uterine infection (oestrus cycle is generally irregular and they may have fever)
• May inject Enrofloxacin, Ciprofloxacin, Ceftriaxone etc. as per body weight. 
• May use anti-inflammatory drug in case of severe inflammation and pain.
Note: Never use intrauterine infusion in acute metritis or when genital discharge is mixed with fresh blood or when 
inflammation is so severe that the cow/buffalo cow shows an arched back posture.
In case of COD—(follicular cyst in this case)
• Manual enucleation: Manual enucleation of cystic structures by palpation per rectum may be done. Complications 
like ovarian haemorrhage and adhesions may follow manual enucleation, so it should be discouraged.
• May inject GnRH hormone. Generally, the cow/buffalo cow will show oestrus 18 to 23 days post treatment.
• May administer HCG: Inject Chorulon: 3000–5000 IU i/v. Cow/buffalo cow generally develops oestrous within 
20 to 30 days post treatment. A second or third dose may be required in few cases.
• May administer progesterone 50 to 100 mg i/m for 14 days or a single dose of 750 to 1000 mg repository 
progesterone.
If due to faulty AI practices
• Animal should not be excited 15 minutes prior to, during or 15 minutes after AI. Excitation causes the release of 
adrenaline thereby lowering the action of Oxytocin which is required for sperm transport.
• Proper thawing of frozen semen and AI should be done within few minutes after thawing.
• Proper insemination technique should be followed to deposit the semen just after the anterior end of cervix or in 
the body of uterus otherwise it will cause traumat o the genital tract.
18 Manual for addressing major reproductive problems and mastitis in dairy animals in Nepal
2.2.4. Advice to the farmers
• Should maintain proper plan of nutrition. Should provide good quality concentrate feed along with addition of 50 
gm of mineral mixture daily. In addition, should provide sufficient good quality green and dry fodder and ad lib 
clean drinking water.
• Should go for AI and in case AI facility is not available, go for natural service with a good quality bull with a 
satisfactory breeding record.
• Should not use a breeding bull for natural service to a cow/buffalo cow that is already infected with genital 
disease and having a history of RB. It will make the bull infected with sexually transmissible disease.
• Should serve the cow/heifer properly during heat. A prolonged heat may create confusion to the attending 
veterinarian and would lead to RB. Should narrate the history properly to the attending veterinarian.
• Should do AI at proper timing. It is the farmer who can give the knowledge on timing ofo estrus.
• Never hide any information on previous history of RB, conception failure, service by infected bull etc. from the 
attending veterinarian. Many farmers think that on giving the true information, the cow/heifer would not be 
inseminated by him/her, rather a course of treatment would be prescribed which would make the conception 
further delayed and costlier.
2.3. Abortion
The expulsion of a dead or live recognizable size of foetus at any stage of gestation (45–60 days onwards to 
parturition) from the uterus is called abortion (Figure 10).
Figure 10. An aborted foetus.
 
Source :https://agritech.tnau.ac.in/expert_system/cattlebuffalo/Abortion.html
Manual for addressing major reproductive problems and mastitis in dairy animals in Nepal 19
2.3.1. Possible causes of abortion
May be of noninfectious or infectious type
Noninfectious (Ahmed 2015; Hovingh 2009):
• Heat stress causes foetal hypotension, hypoxia and acidosis that may lead to abortion.
• High maternal temperature due to pyrexia is more lethal than environmental heat stress.
• Mycotoxins, especially those having estrogenic activity leads to abortion.
• Use of steroids or other drugs contraindicated in pregnancy. Never use steroids during pregnancy. If situation 
calls for use of such drug, use only the pregnancy safe steroids.
• Severe trauma, kicking or hitting with horns by adjacent cows/buffalo cows, falling during transit etc.
• Over exhaustion because of driving pregnant cow/buffalo cow for a long distance on foot, running out of fear or 
being chased by dogs etc. 
• Threatened/habitual abortion—abortion may take place when there is insufficient release of maternal 
progesterone due to a weak CL.
Infectious:
• Brucellosis or Bang’s disease
• Abortion mainly takes place in between 6–9 months of gestation.
• May be accompanied with retention of placenta or RB.
• Campylobacter foetus (previously Vibrio)
• Abortion may take place in between 5–8 months of gestation.
• Rate of abortion may be less than 10%.
• Corynebacterium pyogenes
• Abortion may take place in last trimester of gestation.
• Rate of abortion may go up to 64%.
• Listeria species
• Abortion takes place in 7th month of gestation.
• Rate of abortion may go up to 50%.
• Trichomonas foetus
• It is one of the most common protozoa causing many early embryonic deaths and foetal deaths leading to 
abortion.
• Abortion may take place in 1st half of gestation.
• Leptospirosis
• Abortion may take place in the last trimester of gestation.
• Rate of abortion may be 5–40%.
• Mycotic abortion caused by various species of fungus like—Aspergillus spp etc.
• Abortion may take place from 4 month to full term of pregnancy.
• Most common in winter.
20 Manual for addressing major reproductive problems and mastitis in dairy animals in Nepal
2.3.2. Approach to disease investigation
 Step I: Taking animal history
• Age of animal
• Number of lactations
• Breed of animal
• System of farming (intensive/semi-intensive)
• Month of abortion during the time of gestation
• Appearance of foetus (lathery/dry/fresh)
• Any chronic debilitating diseases suffered by the animal
• Any abnormal symptom shown by animals
• History of any injury/trauma
• History of any stress
• History of abortion in the herd
Step II: Clinical observation
• General health of the animal
Step III: Laboratory investigation
Possible tests
• Milk ring test (MRT)
• Rose Bengal Plate Test (RBPT)
• ELISA/PCR
• Culture and sensitivity test
Sample required:
• MRT—aseptically collected milk sample
• RBPT —blood serum sample
• ELISA/PCR—blood serum sample
• Culture and sensitivity test—vaginal/uterine discharge
Manual for addressing major reproductive problems and mastitis in dairy animals in Nepal 21
Table 5. Differential diagnosis of causes of abortion
Conditions Observations Probability of cause
Stage of gestation Any time Over exhaustion, severe trauma, mycotoxins
First trimester Trichomonas foetus
First and second trimester Mycotic abortion habitual abortion, Listeria spp
Second or third trimester Leptospirosis, Listeria spp, Brucella spp
Third trimester Corynebacterium pyogenes
Season Summer, with higher Heat stress
temperature and humidity
Proximity to horned cow/buffalo cow Severe trauma
Association of retained placenta Yes Brucellosis
Pyrexia Yes High maternal temperature
Transportation Yes Stress due to transportation
History of medication Given Use of steroids or other drugs
2.3.3. Possible line of treatment
Step I: Curative treatment: If placenta is not shredded, may remove the placenta as per procedure described 
in section 2.5 Retained placenta of this protocol.
• May irrigate the uterus with potassium permanganate solution or other antiseptic solution of normal saline or 1–2% 
of potassium iodine solution or normal saline + antibiotic solution. 
• If suspected abortion due to Brucellosis, should not touch the aborted materials and placenta with bare hands. 
Placenta should be removed taking all possible aseptic precautions and it should be disposed off by deep burial 
and blood sample should be collected and sent to veterinary laboratory for confirmatory diagnosis. 
In case of bacterial infection
• Antibiotic injection like—Ceftriaxone and Sulbactum, Amoxycillin and Cloxacillin, Enrofloxacin etc. may be given 
for 3–5 days. Duration of antibiotic therapy depends upon whether there is preexisting infection or it is to prevent 
further infection.
In case of Trichomonadal abortion
• May inject Oxytocin i/v to induce uterine contractions for complete evacuation of uterus.
May perform intrauterine deposition of Levofloxacin, Ornidazole and Alpha Tocopherol combination. Ciprofloxacin 
and Tinidazole combination, Povidone iodine (50:50 in water) etc. may be given daily for 3 days. Povidone iodine is 
highly effective in presence of pus, debris and blood in the uterus while other antimicrobials lose their effectiveness:
• May use Enrofloxacin, Ciprofloxacin, Ceftriaxone etc. parenterally as per body weight.
• May inject vitamins A, D3 and E weekly for three weeks.
• Should allow sexual rest in the subsequent heat.
• May go for AI/natural service in the 3rd heat, followed by injection of 1500 IU HCG immediately after AI/natural 
service.
• May go for oral ecbolic preparations—200 ml on the first day followed by 100 ml for 3 consecutive days. Should 
double the dose for animals weighing above 400 kg. It induces uterine contraction and hastens involution of the 
uterus.
22 Manual for addressing major reproductive problems and mastitis in dairy animals in Nepal
If there is history of threatened/habitual abortion
• May inject progesterone 500–750 mg i/m. First injection on 45th day, followed by 4–5 injections on every 10 
days interval for animals aborting in early pregnancy.
• Or, 2 ml i/m for 3 days in probable calving month, followed by 3 injections at weeklyi ntervals for abortion in mid/
late pregnancy.
Step II: Preventive measures
• Should vaccinate against brucellosis
• Offer good quality feed to pregnant cow/buffalo cow
• Feed contaminated with moulds should not be offered.
2.3.4. Advice to the farmers
• Maintain good hygiene and cleanliness in the farm and surroundings.
• Keep the pregnant cows/buffalo cows a bit away from the other cows/buffalo cows so that there is least 
possibility of injury/trauma from other cows/buffalo cows.
• Avoid slippery floor.
• Arrange for proper air circulation during summer so that heat stress could bem inimized.
• Not to drive the cow/buffalo cow for a long way.
• Not to transport pregnant cows/buffalo cows.
• Keep close contact with the local veterinarian if there is history of threatened/habituala bortion.
• Bury at 6 feet deep all the aborted materials away from the farm/homestead.
• Segregate the affected animals from the others.
• Cull the cow/buffalo cow if infected with brucellosis.
• Be very careful in handling the cow/buffalo cow as well as the aborted materials because brucellosis is one of 
the major causes of retained placenta and brucellosis is a very easily communicable zoonotic disease that can be 
transmitted to the farmer while handling the cow/buffalo cow as well as the aborted materials. It causes undulant 
fever and sterility in humans.
Manual for addressing major reproductive problems and mastitis in dairy animals in Nepal 23
2.4. Stillbirth
Expulsion of dead foetus at the time of parturition is called stillbirth.
2.4.1. Possible causes of stillbirth
• Difficult calving: Trauma caused by technical assistance at delivery such as subcutaneous, subdural or internal 
bleedings and external lesions. Abundant amounts of mucus in the respiratory tract which may be a sign of 
ceased placenta function before the birth process was completed, resulting in suffocation of the calf.
• Intrauterine death: Subcutaneous oedema and hydropsies, dehydration/mummification. The state of 
decomposition as an indicator of how long the calf has been dead in uterus.
• Foetal malformations: Calves with visible malformations.
• Unspecified infections: Calves with unspecified infections (born after normal gestation length).
• Infectious causes: It’s important to rule out infectious causes of stillbirths, such as Neospora caninum, Salmonella 
spp, Listeria monocytogenes, Q fever, Campylobacter foetus venerealis, Chlamydia and Brucella. Mycotoxins have 
also been incriminated.
• Genetic defect.
• Clinically normal at fullterm, unknown cause of death: Clinically normal (wellformed) calves. Lack of external signs 
of difficult calving. No malformations.
2.4.2. Approach to curative and remedial measures: Same as in case of 
abortion.
2.4.3. Advice to the farmers
• Maintain good hygiene and cleanliness in the farm and surroundings.
• Keep the pregnant cows/buffalo cows a bit away from the other cows/buffalo cows so that there is least 
possibility of injury/trauma from other cows/buffalo cows.
• Avoid slippery floor.
• Arrange for proper air circulation during summer so that heat stress could bem inimized.
• Not to drive the cow/buffalo cow for a long way.
• Not to transport pregnant cows/buffalo cows.
• Keep in touch with the local veterinarian if there is history of threatened/habituala bortion.
• Bury the aborted materials away from the farm/homestead.
• Segregate the affected animals from the others.
24 Manual for addressing major reproductive problems and mastitis in dairy animals in Nepal
2.5. Retained placenta
As showed in Figure 11, retained placenta is defined as nonseparation of the placenta/foetal membranes by 24 hours 
after calving. It results from nondetachment of cotyledons from the maternal caruncle.
Figure 11. A case of retained placenta.
Source: Journal of Entomology and Zoology Studies 2018; 6(6): 603-605
2.5.1. Possible causes of retained placenta
• Uterine inertia: Uterine muscles become exhausted after prolonged contraction against an obstructing or 
oversized foetus. There is usually subclinical hypocalcaemia and/or hypoglycaemia.
• Abortion/stillbirth: Abortion caused by brucellosis is almost always followed by retained placenta.
• Advanced age of cow/buffalo cow: Advanced age causes weaker myometrial contractions.
• Dystocia: Dystocia causes exhaustion of uterine muscles as a result of prolonged uterine contraction.
• Vitamins A and E and selenium deficiency: Lack of vitamins A and E along with selenium may lead to reduced 
myometrial tonacity.
• Placentitis: When there is inflammation of the placenta, may be inflicted by injury or infection, the inflamed 
placenta causes retardation/inhibition of detachment of uterine caruncles and placental cotyledons.
2.5.2. Approach to disease investigation 
Step I: Taking animal history
• Age of the cow/buffalo cow
• Breed of the cow/buffalo cow
• Number of lactations
• How long was the labour?
• How long it took to expel the placenta after normal delivery/abortion/stillbirth
• History of abortion/stillbirth if any in the animal and herd
• If aborted, at which stage of gestation
Manual for addressing major reproductive problems and mastitis in dairy animals in Nepal 25
• History of previous treatment if any
• Appetite is normal or off fed
• Milk yield normal or reduced
Step II: Clinical observation
• Colour and smell of the placenta
• Is the placenta moist or dry?
• Body temperature and pulse
• Is there any straining?
• How much portion is hanging out?
2.5.3. Possible line of treatment 
Step I: Medicine
• May inject Oxytocin, 75–100 IU i/m immediately after calving. This may loosen the attachmentb etween the 
cotyledons and caruncles.
• PG F2 α @ 5 ml i/m.
Step II: Manual removal
Procedure of manual removal:
• Following aseptic procedure, grasp the hanging placenta in the right hand (with gloves) and twist it like a rope so 
that the placenta can be more easily managed.
• Introduce the lubricated left hand (with gloves) into the uterus keeping the hand in between the uterus and 
placenta.
• Grasp the individual cotyledon and its caruncle between the thumb and fingers and the two structures 
(cotyledon and caruncle) should be gently separated by rolling, pushing and squeezing motion. This may be 
aided by traction with the right hand.
• Detach all the cotyledons from the caruncles trying first from the cervical area, then from nongravid horn and lastly 
from the gravid horn. Maintain tension on the hanging placenta during the entire procedure and try to twist the 
outer portion.
• Sometimes, especially in exotic and large breeds of cow/buffalo cow, the cranial end of the horn may not be 
accessible to hand, in such case, traction brings the apex of the horn to reach of the hand and makes it possible to 
separate the cotyledons and caruncles.
• Apply some traction to the placenta in a to and from motion u n til the entirep lacenta comes out.
• Be careful not to break any uterine caruncle which may lead to severeh aemorrhage.
• Irrigate the uterus with potassium permanganate solution or other antiseptic solution.
• Insert four numbers of intrauterine preparation containing Nitrofurazone and urea. It may be continued for 3–5 
days in suspension form with the help of a catheter.
26 Manual for addressing major reproductive problems and mastitis in dairy animals in Nepal
• Removal of all the foetal membranes is highly desirable because if any portion is left inside, it will be a potential 
focus of infection.
• If suspected to be due to brucellosis on clinical symptoms and history, utmost aseptic measures must be taken to 
avoid zoonotic transmission and all the biological waste should be properly treated with strong disinfectant and 
then deeply buried.
Step III: Post manual expulsion of placenta
• May give antibiotic injection such as—Ceftriaxone and Sulbactum, Amoxycillin and Cloxacillin, Enrofloxacin etc. 
for 3–5 days. Duration of antibiotic therapy depends on whether there is preexisting infection or it is to prevent 
further infection.
• May inject Siquil @ 5 ml i/m in case of severe straining.
• May give oral ecbolic preparations—200 ml on the first day followed by 100 ml for 3 consecutive days. May 
double the dose for animals weighing above 400 kg. It induces uterine contraction and hastens involution of the 
uterus.
2.5.3. Advice to the farmers
• Be very careful in handling the cow/buffalo cow as well as the aborted materials.
• Never try to pull the hanging portion of placenta which will cause removal of only the visible external portion and 
the remaining portion may remain inside and may cause severei nfection and even septicaemia. Pulling with force 
may cause uterine prolapse.
• May use oral ecbolic preparations right after calving on advice of the local veterinarian.
• Should not wait for too long, may call the nearby veterinarian/paraveterinarian for removal of placenta.
• Maintain a proper plan of nutrition. 
• Bury the placenta away from the farm/homestead.
• Cull the cow/buffalo cow if infected with brucellosis.
2.6. Purulent vaginal discharge
Occasionally, there may be discharges coming out through vagina of a cow/buffalo cow at any point of time. Though 
it reflects disease inside the vagina, cervix or the uterus, it is not always pathological. Vaginal discharge may be of 
different types which are indicative of different condition of the female genital tract.
2.6.1. Vaginal discharges and possible causes
• Transparent and moderately viscous extending to ground without breach—- mayi ndicate that the cow/buffalo cow 
is in heat. It should be correlated with other symptoms.
• Transparent and watery—may indic ate that  the cow/buffalo cow is in early heat. Should be correlated with 
others ymptoms.
• Transparent and viscous, cuts off in the midway—may indicate that the cow/buffalo cow is in late heat. Should be 
correlated with other symptoms.
Manual for addressing major reproductive problems and mastitis in dairy animals in Nepal 27
• Profuse watery—may indicate mucometra.
• Cloudy and with variable degree of consistency—may indicate infection in the vagina, cervix, or uterus.
• White or whitish-yellow mucopurulent vaginal discharge comes out when a diseased cow/buffalo cow sits 
down—may indicate severe endometritis.
• Transparent and viscous in late pregnancy—may indicate calving is nearing. Should be correlated with other 
symptoms.
• Light yellowish green vaginal discharge—may indicate foetal anoxia with associated expulsion of meconium. 
Should be correlated with other symptoms.
• Frequent and copious vaginal discharge than normal, opaque or whitish grey in colour- may indicate follicular 
cyst.
• Bloody—may indicate acute and severe endometritis.
Diagnosis cannot be done solely on the indications given for vaginal discharge. Correlating with other clinical 
findings are essential. For confirmation, vaginal discharge should be collected for testing in the Disease Diagnostic 
Laboratories.
2.6.2. Possible line of treatment
Step I: Curative treatment
• Treatment should be given as per the primary condition/disease which is to bec onfirmed in accordance with 
other symptoms and history which are given above.
• Give further treatment if the laboratory report is different from what was diagnosedt entatively.
Step II: Advice to the farmer
• Not to touch the discharge with bare hand.
• Isolate the cow/buffalo cow, if possible. If there is space for isolation, keep the cow/buffalo cow a bit away from 
the others.
• Disinfect the floor with a good disinfectant like phenyl, potassium permanganate solution etc.
• Consult with the nearby veterinarian/paraveterinarian.
• Never consider the case as the cow/buffalo cow to be in heat and go for natural service or AI.
2.7. Dystocia
When the stage I or especially, the stage I labour is markedly prolonged, i.e. more than an hour and becomes difficult 
or impossible for the dam to expel out the foetus without artificial aid, the condition is called dystocia.
2.7.1. Possible causes of dystocia
Foetal factors: As showed in Figures 12 to 16, developmental defect like conjoined twins, hydrocephalus, foetal 
monsters, foetal death, abnormal foetal position, abnormal foetal posture, abnormal foetal presentation, foetal 
oversize, foetal emphysema etc.
28 Manual for addressing major reproductive problems and mastitis in dairy animals in Nepal
Figure 12. Developmental defect: Hydrocephalic                                  Figure 13. Developmental defect: Conjoined twin. 
foetus removed by foetotomy
Source: Buffalo Bulletin, 2007, 26(123)
Figure 14. Dicephalic monster.
Source: Indian Journal of Field Veterinarians (The), 2011. 7(1), pp.72-74.
Figure 15. Abnormal foetal presentation:          Figure 16. Abnormal foetal posture:  
Breech presentation            Bilateral shoulder flexion
Source: https://www.slideshare.net/MohamedWahab2/5th-year-practical-revision-fetal-presentations
Manual for addressing major reproductive problems and mastitis in dairy animals in Nepal 29
Maternal factors:
• Incomplete cervical dilation—sometimes the cervix fails to dilate properly during calvingc ausing dystocia.
• Uterine torsion—it is one of the most common cause of dystocia in buffalo than cow/buffalo cow in Nepal.
• A disproportionate size of the foetus in relation to the size of the pelvis.
• Metabolic deficiency—hypocalcaemia, hypoglycaemia, hypomagnesaemia, obesity, oxytocin deficiency.
• Secondary uterine inertia—uterine muscles become exhausted after prolonged contraction against an obstructing 
or oversized foetus. There is usually subclinical hypocalcaemia and/or hypoglycaemia.
• Ectopic pregnancy—when the fertilized ovum is implanted in any tissue other than the uterine wall.
• Inadequate pelvis size—sometimes the pelvic girdle fails to expand during calving, or the pelvic girdle is too 
narrow to allow the foetus to be expelled under the influence of uterine contractions.
• Hip joint fracture/hip dysplacia—it causes obstruction to the birth canal and surgical intervention is required.
• Birth passage is blocked with neoplastic growth.
2.7.2. Approach to disease investigation
Step I: Taking animal history
Cases of dystocia should be attended without any delay.
• A thorough history regarding length of gestation, parity and problems seen during pregnancy should be 
investigated. Should find out when the animal started straining and if any water or fluid bags have appeared and/
or ruptured. Also, should take history of previous hip dislocation/fracture.
• It is important to understand what has been done so far by the farmer including any drugs administered.
Step II: Physical examination 
Physically examine of the dam who might have systemic illness, such as milk fever. Proper restraint and assistance 
should be sought and initial treatment may include fluid therapy, pain relief and the provision of calcium.
Step III: Vaginal examination
The vulva, vagina and cervix should be assessed for dilation, torsion or injury. Copious lubrication should be used at 
all stages of the examination.
Step IV: Foetal assessment should establish its presentation, posture and position and identify any palpable 
structures. Foetal viability can be assessed by feeling pulses on the limbs or tongue.
2.7.3. Possible line of treatment/correction
Anaesthesia is not essential, but an epidural can be administered to help reduce straining.
Manual correction of dystocia: It relies on traction to facilitate expulsion, repulsion of the foetus back into the 
uterine lumen to enable further manipulation.
30 Manual for addressing major reproductive problems and mastitis in dairy animals in Nepal
• Use of lubricants is essential. Should apply lubricant liberally, especially in case of prolonged operations, two or 
more times or frequently so that the foetus or birth-canal does not become dry.
• Ropes can be applied to both legs and the head and traction applied while manually dilating the vagina and 
vulva.
• If the foetus is too large to be delivered vaginally, a caesarean operation or a foetotomy should be performed.
Possible treatments for the dam include:
• Analgesics and anti-inflammatory drugs—it can be given at the start. Short acting steroids can help reduce 
swelling and oedema. If cervix is not dilated properly use PGF2alpha along with valethamate bromide injection 
I/V or IM rout. 
• Antibiotics like Ceftriaxone, Amoxycillin and Cloxacillin, Ceftiofur sodium, Enrofloxacin etc. should be given for 
5–7 days after calving due to the increased risk of complications such as retained placenta and metritis.
Step II: Prevention
Foetomaternal disproportion is one of the major contributors to dystocia and this can be prevented with proper 
reproductive management.
Postpartum complications
Dystocia leads to an increased risk of postpartum complications in cattle, such as:
• Retained placenta: usually treated by manual removal
• Metritis: which can lead to toxaemia and laminitis and should be treated aggressively
• Uterine prolapse
• Cervical tears
• Uterine rupture
• Uterine haemorrhage
• Perineal lacerations.
2.8. Mastitis
Mastitis is inflaming the udder of a cow/buffalo cow mostly caused by bacterial infection and sometimes by 
other pathogens. These micro-organisms produce toxins which directly damage milk producing tissue of the 
mammary gland. This damage initiates inflammation process within the mammary tissue to eliminate the invading 
micro-organisms. The inflammation contributes to decreased milk production and is primarily responsible for the 
compositional changes observed in milk from infected quarters and cows/buffalo cows (Figure 17).
Manual for addressing major reproductive problems and mastitis in dairy animals in Nepal 31
Figure 17. Mastitis.
Source: Chanduri Nagendra. https://curofy.com/discussion/mastitis-heifer-27cd97e6710e6a3d73f3277cadcb33ff.
There are mainly two types of mastitis—(i) clinical mastitis in which the affected cow/buffalo cow exhibits the clinical 
signs and (ii) subclinical mastitis in which the cow/buffalo cow does not exhibit any visible/clinical signs and the 
disease goes unnoticed.
Clinical mastitis: is characterized by sudden onset, alterations of milk composition and appearance, decreased milk 
production and the presence of the cardinal signs of inflammation in infected mammary quarters of the cow/buffalo 
cow and the disease becomes noticeable.
Subclinical mastitis: no visible signs are seen either on the udder or in the milk, but the milk production decreases 
and the somatic cell count (SCC) increases. It is more common and has serious impact in older lactating animals than 
in first lactation heifers. Because of absence of external manifestation, the diagnosis of subclinical mastitis is difficult. 
It can be diagnosed by milk test either with Mastrip paper (available in the pocket of Mastilep ointment) or by using 
CMT. This is a very simple test that the veterinarian/farmer can perform in the field provided the CMT solution is 
available. 
2.8.1. Possible causes of mastitis
The main causes for developing mastitis are:
• Bacterial infection
• Species of bacteria such as—Staphylococcus aureus, E. coli, Klebsiella spp, Streptococcus spp, 
Corynebacterium spp, coliforms, Arcanobacterium pyogenes etc. may cause mastitis in cows/buffalo 
cows (Jost and Billington 2005).
• About 90% of pathogens responsible for mastitis are found in the cow–barne nvironment.
• Except Arcanobacterium pyogenes, the etiological agent of clinical mastitis, all other micro-organisms 
cause both clinical and subclinical form of mastitis.
• S. aureus, E. coli and Klebsiella spp cause the greatest losses of milk production in primiparous cows/
buffalo cows with mastitis.
• Streptococcus spp, S. aureus, Arcanobacterium pyogenes, E. coli and Klebsiella spp cause most 
significant losses in older cows/buffalo cows.
• Apart from causing mastitis, E. coli spp can lead to severe systemic disorder.
• Fungal (mycotic) infection
• Increased incidence of udder mycosis results from mineral–vitamins deficiencies, anti-oxidant deficiencies, 
imbalanced diet, poor environmental conditions and even weather changes.
32 Manual for addressing major reproductive problems and mastitis in dairy animals in Nepal
• In general, fungi are rare cause of mastitis but sometimes can occur in epizootic proportions.
• Usually in farms with poor environmental and hygienic conditions, poor quality of materials used as 
bedding (e.g. straw) with high humidity can be source of fungi causing mastitis in cattle.
• Fungi are also reason of udder inflammation when udder is washed with water but is not dried.
• Several species of yeast may cause bovine mastitis (Candida, Trichosporon, Saccharomyces and 
Rhodotorula).
• Algae (Protothecal)—Mastitis can also be caused by algae (organisms from Prototheca genus) in a lesser 
proportion. Poor environmental conditions, inappropriate milking hygiene and prolonged antibiotic therapy can 
be the reason of increased protothecal mastitis occurrence.
• Trauma—Inflammation of the udder may be caused by various forms of trauma such as—friction with a very rough 
floor, threshing by hoof of other cow/buffalo cow, injury while trying to jump over a fence or concrete wall (as in a 
manger), fisting type of milking practice or even a polyp developed inside the teat canal.
• Injudicious use of antibiotics—It is a general practice of the farmers to discontinue thec ourse of antibiotics 
against mastitis prescribed by the veterinarian. They just stop it on seeingt he disappearance of the visible 
symptoms. This is one of the causes of relapse of the disease and subclinical form of the disease. They also use 
antibiotics at their own choice without having a prescription from a registered veterinarian. This kind of practice 
has influence on increased incidence of mycotic bovine mastitis. Eliminating bacterial flora antagonist to fungal 
growth during antibiotic therapy stimulates fungal multiplication. It also develops bacterial resistance to the 
antibiotic given.
Sequels of mastitis:
• One of the most detrimental sequels of mastitis is fibrotic changes that take place in the mammary gland. In such 
case, the functional milk alveoli are replaced with fibrous tissue as a result of severe inflammation of the gland and 
despite the cow/buffalo cow being totally free from the infective cause, the cow/buffalo cow will be no more in a 
position to givea  good milk yield.
• Another one is damage done to the mammary gland and, even to the body system, by the toxins produced by 
the pathogens which are not destroyed by antimicrobial drugs.
2.8.2. Approach to disease investigation 
Step I: Taking animal history
• Duration of suffering/showing symptoms
• History of mastitis in previous lactation
• Has any medication given already and if so, the details of the same?
• History of prepartum udder oedema
• History of practicing insertion of grass stem into teat canal prior to calving (a malpractice adopted in many places 
of Assam) to get the cow/buffalo cow acquainted tom ilking
• Use of Oxytocin in previous lactation
• Blocked teat canal of any quarter.
Manual for addressing major reproductive problems and mastitis in dairy animals in Nepal 33
Step II: Clinical observation of the animals Observing for clinical findings:
In clinical mastitis:
• All the cardinal signs of inflammation—swelling, pain, redness and hotness (rubor, dolour, calor, tumour) of the 
udder and also, hardness; rise in body temperature
• Oedema of the udder extending to the naval
• Drop in milk yield
• Pus/blood/flakes in milk
• Offensive odour of milk
• Curdled milk
• Watery milk
• Hard milking causing reduced flow of milk
• Change in pH of milk
• A reduction in mobility, due to the pain of a swollen udder or simply due to feeling unwell and lack of appetite
• In severe cases of acute, clinical mastitis, the cow/buffalo cow may appear very ill.
In subclinical mastitis:
A cow/buffalo with subclinical mastitis may appear otherwise normal with the following signs—
• A reduction in yield potential
• A high SCC
• Milk shows flakes like or powdery residues on straining
• A hard, fibrotic appearance of the udder (any of the quarters)
• Positive result of CMT
• Change in pH of milk on pH strip test
• Milk gets sour earlier than normal, i.e. before 4 hours of milking.
Step III: Laboratory Investigating possible tests
• CMT (rapid test at farm level) (Figures 18 and 19).
34 Manual for addressing major reproductive problems and mastitis in dairy animals in Nepal
Figure 18. A CMT kit.                 Figure 19. Interpreting results from CMT.
• Isolating and identifying causative agent (bacteria)
• SCC
Sample required:
• CMT—Aseptically collected milk sample
• Isolating and identifying causative agent—Aseptically collected milk sample
• SCC—Aseptically collected milk sample
2.8.3. Possible line of treatment 
Step I: Curative treatment
• May give conservative treatment with a course of suitable antibiotic.
• May give an anti-inflammatory drug.
• May use corticosteroid drugs in case of sever degree of inflammation.
• May use proteolytic enzymes like—serratiopeptidase, chymotrypsin etc. to heal the inflammatory tissues.
• May infuse intramammary preparations as per schedule.
• May give hot fomentation with saturated magnesium sulphate solution.
• May use externally applicable gels etc.
• In case of systemic involvement, may use DNS infusion.
• May surgically remove obstructing teat polyp, if any.
• May drain out the infected quarter with antiseptic solution by using milk siphon.
Manual for addressing major reproductive problems and mastitis in dairy animals in Nepal 35
2.8.4. Advice to the farmers
• Strictly maintain hygiene in the farm.
• Strictly maintain personal hygiene.
• Handle and milk the affected cow/buffalo cow only after completing milking the other cows/buffalo cows in the 
farm.
• Never practice fisting method of milking.
• Never use oil etc. to make the teats slippery during milking.
• Separate the affected cow/buffalo cow from the herd.
• Complete the course of treatment, especially that of antibiotics, as prescribed by the attending veterinarian.
• Never use the milk for sale or consumption until the withdrawal period is over.
• Never drip the milk in the floor.
• Dip teats in the antiseptic solution (Potassium iodine 90 ml + glycerine 10 ml) after completing the milking.
•  Provide green fodder just after milking so that cow/buffalo cow stand until the teat canal closed.
• To check the pH of milk periodically using pH paper strip for detecting subclinical mastitis.
36 Manual for addressing major reproductive problems and mastitis in dairy animals in Nepal
3. Process to be followed for per rectal 
examination
3.1. Materials required for rectal examining a cow/buffalo cow
• Full sleeve hand gloves
• Face mask
• Apron
• Shoe cover
• Soap
• Hand sanitizer
• Lubricant e.g. coconut oil, petroleum jelly etc. (Figures 20 and 21). 
Figure 20. Per rectal exploring a cow/buffalo cow.  
Figure 21. Materials required for rectal examining a cow/buffalo cow.
Full sleeve hand glove    Face mask     Apron
Manual for addressing major reproductive problems and mastitis in dairy animals in Nepal 37
3.2. Personal precaution during rectal examination
• Wash the hands with soap and water
• Use disposable gloves, apron, mask and shoe cover
• After examining, dispose the personal protective clothing, especially glovesh ygienically (burn/deep burial)
• Wash your hands with soap water
• Wash your cloth after returning to residence.
3.3. Procedure for rectal examination
• After wearing gloves, lubricate the left hand (in case of right handed person) and insert into the rectum very 
gently;
• The animal may put some opposing force which is due to peristaltic movement of the bowel and wait until it 
subsides;
• Push the hand forward and feel the uterine fundus;
• Go further and feel the horns and then the ovaries;
• Explore all the organs one by one, i.e. ovaries, horns, fundus of the uterus, os cervix and vaginal canal;
• Gently withdraw the hand;
• Extreme care should be taken so that the rectal mucosa is not torn or damaged.
38 Manual for addressing major reproductive problems and mastitis in dairy animals in Nepal
4. Sample collecting, labelling, storing and 
dispatching
Different types of samples are to be collected for different types of tests following specific procedures and then it 
should be packed, preserved if required and finally, to be dispatched to the laboratory or used on the spot for the 
rapid tests.
4.1. Steps in collecting samples to dispatching to laboratory:
• Collecting biological sample (discharges, milk, faeces etc.) aseptically followings tandard protocol;
• Transporting to the hospital in cool temperature using cool box/thermosflask;
• Packaging and dispatching to laboratory following standard protocol as early asp ossible in thermocol box 
adding ice pack/gel pack.
4.2. Procedure for collecting vaginal/uterine discharge:
4.2.1. Materials required:
• Uterine catheter,
• Adaptor,
• AI gun,
• AI sheath,
• Pipette,
• Cotton gauze,
• Syringe,
• Sterilized container/vial,
• Isotonic saline solution.
4.2.2. Methods
4.2.2.1. By back racking
• Put on a glove on the left hand and properly lubricate it;
• Insert the left hand into rectum, lift the uterus and cervix upward and massage ina  backward direction;
Manual for addressing major reproductive problems and mastitis in dairy animals in Nepal 39
• Continue to massage backwardly to the vagina through the rectal wall which willa llow cervical mucus to flow 
down through the vulvar lips;
• Collect the discharge in a wide mouthed sterilized container/vial;
• Cut the discharge with the help of scissors if it remains hanging.
4.2.2.2. By using uterine catheter
• Put on a glove on the left hand and properly lubricate it;
• Insert the left hand into rectum, hold the cervix and uterus in the grasp of theh and;
• Open the vulvar lips and introduce the uterine catheter with the right hand into vagina (anterior part) or cervix or 
from where genital discharge must be collected;
• Fit a syringe to the outer end of the uterine catheter by the right hand and aspirate the mucus;
• Collect the discharge in a wide mouthed sterilized container/vial.
4.2.2.3. By using AI gun and AI sheath: (It is done in case the uterine catheter is not readily 
available)
• Put on a glove on the left hand and properly lubricate it;
• Insert the left hand into rectum, hold the cervix and uterus in the grasp of the hand cervix;
• Open the vulvar lips and introduce the AI gun with the right hand, with the AI sheath fitted, into vagina (anterior 
part) or cervix or from where genital discharge must be collected;
• Withdraw the AI gun leaving the AI sheath in place within the grasp of the left hand;
• Trim the slit outer end of the sheath with scissors to facilitate fitting of the syringe;
• Fit a syringe to the outer end of the AI sheath by the right hand and aspirate the mucus;
• Collect the discharge in a wide mouthed sterilized container/vial.
4.2.2.4. ampon method: (not a common practice)
• Take a sterile cotton gauze tampon of about 1 g and attach a string to it;
• Put on a glove on the left hand and properly lubricate it;
• Insert the sterilized gauze tampon into the vagina using the left hand;
• Leave the tampon in the vagina for 20 minutes;
• Remove the tampon from the vagina by pulling the string;
• Place the tampon in a sterilized bottle containing isotonic saline solution.
4.3. Procedure for collecting milk sample:
The veterinarian should collect milk samples adopting the following procedure for on the spot testing such as CMT 
kit, pH metre strip, SCC etc. or for onward transmission to diagnostic laboratories for identifying pathogen, culture 
and sensitivity test, antibiogram etc.
40 Manual for addressing major reproductive problems and mastitis in dairy animals in Nepal
4.3.1. Materials required:
• For collecting sample directly from the cow/buffalo cow, take Latex or nitrile gloves (one pair per cow/buffalo 
cow), predip, paper or cloth towels (one per cow/buffalo cow), cotton balls soaked in 70% rubbing alcohol, 60 
ml or larger sterile screw on vials (one for each quarter).
• A cool box/thermosflask to carry the sample;
• Required ice/gel packs in cool box to keep the sample cool during transportation;
• Personal protective clothing like apron, gloves, mask etc.;
• Sticker tags, marker, notepad, mask, sanitizers and biohazard bag;
• Disposal bag for carrying disposable materials like leftover milk, gloves, mask etc.;
• Spirit lamp/alcohol for sterilizing the instruments on the spot.
4.3.2. Procedure for collecting milk sample directly from the udder of 
cow/buffalo cow
• Ask the owner of the cow/buffalo cow to clean the udder and teats of the cow/buffalo cow thoroughly with water 
(Figure 22).
• Put on the clean gloves, apply predip to teats that have already been washed of manure, dirt, or sand.
• Strip two to three streams of milk from each teat to flush the teat canal and thereby to reduce contamination risk.
• Dry teats thoroughly with an individual cloth towel, paying close attention particularly to the teat end.
Figure 22. Clean udder.
• While holding the top of the teat steady, wipe the end of the teat well with an alcohol soaked cotton ball (Figure 
23). Use as many cotton balls as necessary until the cotton ball still looks clean after using.
Manual for addressing major reproductive problems and mastitis in dairy animals in Nepal 41
Figure 23. Cleaning of the teat.
• Open the milk vial and immediately take the sample, making sure not to touch the inside of the tube or bottom 
part of the lid. Hold the milk vial about 3 inches from the teat end and fill the tube half to three-quarters full of milk. 
Hold the vial at a 45 degree angle to prevent dirt from falling into the vial as presented in Figure 24. 
Figure 24. Collecting milk sample in a sterile milk vial.
• Close the lid immediately and label the top with the date, cow/buffalo cow number and quarter sampled (Figure 
25).
Figure 25. Milk sample.
• Cool samples immediately. Milk samples can be refrigerated for up to 2 days and can be frozen for up to 6 
months. If mailing samples into a laboratory, mail them overnight on ice packs.
• If samples are intended for SCC evaluation, never freeze the samples because the cells will burst and you will get 
a false reading.
Note: Do not place the teat inside the vial while collecting samples.
42 Manual for addressing major reproductive problems and mastitis in dairy animals in Nepal
4.4. Procedure for collecting sample for PCR:
• Tissues/organs: these samples, e.g. liver, lung etc. should be sent whole in individual tubes.
• Faeces: two faecal samples per animal are required. Faecal samples may be pooled, however only up to five 
different samples should be pooled to make up one sample for testing.
• Blood: Aseptically draw 10 ml of blood from the jugular vein and put in a sterile tube with EDTA (purple top 
tubes), other coagulants such as heparin or sodium citrate should not be used.
• Blood samples collected for PCR should be frozen, preferably at –8°C, immediately after collection and 
transported in ice (local transport) or in dry ice (interstate transport).
4.4.1. General requirement for collecting sample:
Make arrangement of all necessary materials before going to sample collection.
• Take a cool box/thermosflask to carry the sample;
• Take required ice/gel packs in cool box to keep the sample cool duringt ransporting;
• Take personal protective clothing like apron, gloves, mask etc.;
• Take sticker tags, marker, notepad, mask, sanitizers and biohazard bag;
• Take a disposal bag for carrying disposable materials like leftover milk, gloves,m ask etc.;
• Take a spirit lamp/alcohol for sterilizing the instruments on the spot;
• For collecting sample directly from the cow/buffalo cow: Take Latex or nitrile gloves (one pair per cow/buffalo 
cow), predip, paper or cloth towels (one per cow/buffalo cow), cotton balls soaked in 70% rubbing alcohol, 
permanent marker, 60 ml or larger sterile screw on vials (one for each quarter).
4.4.2. Information to be submitted along with the sample:
• Type of the sample (e.g. milk/whole blood/blood serum/faeces etc.);
• Species: Cow/buffalo;
• Type of animal: Exotic/crossbreed/nondescript;
• Quarter of udder: L (left) / R (right) / F (fore) / R (rear);
• Sample number;
• Weight/volume of the sample;
• Place of collection;
• Date and time of collection;
• Name of the collector;
• Purpose of collecting the samples (e.g. culture and sensitivity test, RBPT, milkp rogesterone etc.);
• Name, address and signature or thumb impression of the person from whom thes ample has been taken.
Manual for addressing major reproductive problems and mastitis in dairy animals in Nepal 43
4.5. General considerations in sample collecting, handling and 
storage:
• The samples should never be touched with bare hands. Gloves and mask shoulda lways be used in the process 
of collection.
• Outer and inner surface of the container should be cleaned thoroughly.
• Sample should not be allowed to expose to dirty materials/environment afterc ollection and should not be 
mixed with other biological samples.
• Temperature and pH shall be recorded at the collection stage and aftert ransporting to the laboratory.
• Gloves, mask and other materials in contact with the sample must be disposedp roperly.
• The stopper/cover of the container shall be securely fastened to prevent leakageo f the contents in transit.
• The container shall be completely wrapped in strong thick paper.
• The ends of the paper shall be neatly folded in and affixed by means of gum ora nother adhesive.
• Always collect the samples as fast as possible.
4.6.Sample labelling and storing:
• Separate sterile container should be used for each sample.
• Collected sample should be distinguished from other samples by writing the details of the sample on the body 
of the container by a good quality marker and on a sticker tag and pasting the same on the container immediately 
after collection.
• The detail information of the sample should be written in a piece of paper and should put the same in a polythene 
zip bag and stick it at the body of the container by a transparent adhesive tape.
• The labelled container should immediately be transferred to the cool box/thermosflask filled with ice packs.
• The collected container shall be properly secured and sealed so that no tempering is possible after collection. To 
ensure this, signature of the milk producer/trader/sweet maker and a witness should be taken on the sealed pack.
• All samples should be transported to the laboratory by maintaining the cold chain in a cool box/thermosflask with 
gel/ice packs.
• No personal details of the owner of the sample should be supplied to the laboratory technicians who conduct the 
tests to avoid any potential prejudgement by the laboratory technicians.
• The sample should be stored at 4°C and processing should be made immediately.
In case of delay, samples should be refrigerated at 4°C and processing should be done within 96 hours of sampling. 
If sample needs to be preserved for more than 96 hours, it should be stored at –20°C.
44 Manual for addressing major reproductive problems and mastitis in dairy animals in Nepal
4.7. Packaging and dispatching samples to other laboratories:
• For dispatching the samples to other laboratories, sample with their details shouldb e put in a thermocol box. 
Adequate quantity of cool ice pack/gel pack should be put in the box to keep the sample cool during the time of 
transportation.
• The outside of the thermocol box should be wrapped up with white paper and address of ‘From’ and ‘To’ should 
be clearly written on it preferably in all capital letters.
• A certificate should also be enclosed with the box stating the nature of the materials and purpose of sending.
• Packages should be marked clearly to provide information about the contents of package and nature of the 
hazard, if any.
• Sample should be sent by the mode of transportation that can deliver the sample at the quickest possible time in 
the destination. If the transportation time takes more, the ice/gel pack may come to normal temperature and the 
sample may get spoiled.
• The thermocol box should also be marked with ‘Handle with care’ an ‘arrow mark’ showing upside of the box, to 
guide the handlers during transportation.
Manual for addressing major reproductive problems and mastitis in dairy animals in Nepal 45
Annex I. Animal observation check list
Name of the owner/s 
address:
Contact Number
A History Particulars Remarks
 Identification number/name of the 
cow/heifer
Breed
Age
Type Cow/heifer
Number of lactations
Age at first heat
Type of mating Natural/AI
Number of AI required per 
conception
Date of AI/natural mating
Months of gestation, if conceived
Interval between two to three 
successive heats, if not conceived
Age at first calving
Time interval between last calving 
to next heat
Time interval between last calving 
to next conception
History of previous diseases/ Delayed postpartum heat: How long it 
conditions took?
RB:
How many times repeated?
Dystocia: Foetus dead or alive
Retained placenta: How many times?
Abortion: In which month of gestation?
How many times?
Stillbirth: How many times?
Mastitis: In how many previous lactations? 
How many quarters of teat affected?
Is there any blockage of teat canal?
Is there any fibrotic change in the udder?
History of hip dislocation/fracture
46 Manual for addressing major reproductive problems and mastitis in dairy animals in Nepal
Duration of the existing disease/
condition
Previous treatment if any
Vaccination Disease
Date
Deworming Date
Nutritional plan Concentrate feed/conventional feed
Mineral and vitamins supplement Given/not given
B External appearance
General appearance Dull/alert
Body coat Rough/shiny
General health condition Normal/emaciated
Body coat Rough/shiny
General health condition Normal/emaciated
Appetite Normal/off fed
Eyes Dull/bright
C Physical examination
Body temperature °C
Presence of injury in the abdomen Yes/no
Presence of external parasite Yes/no
Presence of worms in manure Yes/no
Appearance of udder Normal/swollen/oedematous/hard/soft
Presence of injury on udder Yes/no
Hanging out of placenta Partial/not visible
Position of foetus Half delivered/not visible
Water bag Not visible/coming out, intact/ruptured
Genital discharge Clear/cloudy/watery/with pus/bloody
Colour of visible mucous Normal/pale/reddish
membrane of eye
D Predisposing factors
Occurrence of disease in Yes/no 
neighbourhood/in the same herd If yes, what kind of disease?
Number of cows/heifers affected
Number of households affected
Scarcity of food Yes/no
Flood Yes/no
Type of floor Rough/damp/waterlogged/earthen
Unhygienic farmstead Yes/no
E Rapid tests done
CMT Results:
MRT Results:
RBPT Results:
pH paper strip test Results:
Ubio Quickvet bovine pregnancy Results:
spot assay
Faecal sample for presence of Results:
worms
Manual for addressing major reproductive problems and mastitis in dairy animals in Nepal 47
Pregnancy diagnosis by Results:
Rectogenital palpation
Pregnancy diagnosis by Doppler Results:
ultrasonography machine
F Laboratory test required if any
Vaginal/uterine discharge: culture Results:
and sensitivity test
Milk: culture and sensitivity test Results:
ELISA/PCR for brucellosis Results:
ELISA test for estimating milk Results:
progesterone concentrations
SCC Results:
48 Manual for addressing major reproductive problems and mastitis in dairy animals in Nepal
References
Ahmed, T. 2015. Abortion in cattle. MSD Veterinary Manual.
Amatya, N., Rasali, D.P. and Rana, R.S. 2000. Evaluation of phenotypic and production characteristics of indigenous 
buffalo types in the western hills of Nepal. Lumle Technical Paper, (2000/1).
Farin, P.W., Youngquist, R.S., Parfet, J.R. and Garverick, H.A. 1992. Diagnosis of luteal and follicular ovarian cysts by 
palpation per rectum and linear array ultrasonography in dairy cows. Journal of the American Veterinary Medical 
Association 200(8): 1085–1089. (https://www.ncbi.nlm.nih.gov/pubmed/1607312)
Hovingh, E. 2009. Abortions in dairy cattle II: Diagnosing and preventing abortion problems.
Jost, B.H. and Billington, S.J. 2005. Arcanobacterium pyogenes: molecular pathogenesis of an animal opportunist. 
Antonie Van Leeuwenhoek 88(2): 87–102.
Kumar, P. 2013. Applied veterinary gynaecology and obstetrics. Lucknow, India: International Book Distributing 
Co.
Lamsal, S., Subedi, D. and Kaphle, K. 2020. Buffaloes Production and Reproduction Efficiencies as Reviewed for 
Parity in Nepal. International Journal of Applied Sciences and Biotechnology 8(1), pp.1-6.
MoALD. 2020. Statistical Information on Nepalese Agriculture 2020/21; Ministry of Agriculture and Livestock 
Development: Kathmandu, Nepal: pp. 3–4.
Neopane, S.P. 2006. Characterization of indigenous animal genetic resources of Nepal. Proceeding of the 6th 
National Worshop on Livestock and Fisheries Research. Nepal Agricultural Research Council, pp.1-11
NDDB. 2021. Study on database of dairy value chain. National Dairy Development Board, Hariharbhawan, Lalitpur, 
Nepal.
Pokhrel, P.K., Kuwar, B.S., Shrestha, N.P., Neopane, S.P. and Shrestha, H.R. 1998. Identification, characterization 
and conservation strategy of Gaddi buffalo. In Proceeding of the 4th Global Conference of Domestic Animal 
Genetic Resources (pp. 77-80).
Rasali, D.P. 1997. Present status of indigenous buffalo genetic resources in the western hills of Nepal. Proceedings of 
the Fourth Global Conference on Conservation of Domestic Animal Genetic Resources. Rare Breeds International: 
168-170.
Rasali, D.P., Joshi, H.D., Patel, R.K and Harding, A.H. 1998. Phenotipic clusters and karyotypes of indigenous 
buffaloes in the western hills of Nepal. Lumle Agricultural Research Centre. Technical Paper, 98/2: 24.
65
ISBN: 92–9146–760–x
Manual for addressing major 
INITIATIVE ON
Sustainable Animal 
Productivity reproductive problems and 
 mastitis in dairy animals 
CGIAR’s Sustainable Animal Productivity for Livelihoods, 
Nutrition and Gender inclusion (SAPLING) is working 
in seven countries focusing on livestock value chains to in Nepal  zones
package and scale out trie d-and-tested, as well as new, 
innovations in livestock health, genetics, feed and market systems. SAPLING aims to demonstrate that improvements in 
livestock productivity can offer a triple win: generating improved livelihoods and nutritional outcomes; contributing to 
women’s empowerment; and, reducing impacts on climate and the environment. Its seven focus countries are Ethiopia, 
Kenya, Mali, Nepal, Tanzania, Uganda and Vietnam. 
ILRI Research Report 29
I L R I   M A N U A L