Superovulatory Response and Progesterone Profile in Murrah Buffaloes (Bubalus bubalis) Pretreated with GnRH Agonist

Buffalo (Bubalus bubalis) is an important livestock of Southeast Asia due to its inherent milk quality (higher solids compared to cattle), higher parasitic resistance, lesser reproductive and mammary infections and higher feed conversion efficiency with low maintenance compared to cattle thereby contributing towards daily economy for marginal livestock farmers (Colli et al., 2018). India is the major mainstay of buffalo breeds contributing about 57.3% of world population (FAOSTAT, 2010). This population has increased but only marginally to less than 1% during last 5-7 years (DADF, 2019) alarming poor reproductive performance due to increased global warming and poor nutritional availability (FAOSTAT, 2010).
       
Elite buffaloes producing 3500-4000 kg milk per lactation, their number is very less, which urgently required to be multiplied by using mutiple ovulation embryo transfer technology (MOET; Mohammed, 2018). MOET has been applied for genetic improvement of livestock with varying level of success. However, the success rate is much lower in buffaloes due to their inherent lower fertility and poor superovulatory response. Variability in the number of ovulation, number and quality of recovered embryo during superovulation is a major limiting factor in the successful implementation of embryo transfer in buffalo (Misra and Tyagi, 2007). The progesterone levels at initiation of superovulatory treatment (Misra et al., 2000) and the effect of dominant follicle (Singh et al., 2015) are largely responsible for variability in the superovulatory response in buffalo. Higher progesterone concentration at the start of superstimulatory treatment has positive effect on the subsequent ovulatory response in buffalo (Heleil and El-Deeb, 2010).
       
During MOET, pretreatment with GnRH agonist negate the adverse effect of dominant follicle and improve superovulatory response in cattle. There are little known researches about the endocrine factors that affect the ovarian response and embryo production in superovulated buffalo pretreated with GnRH agonist. Therefore, the objective of present study was to evaluate the serum progesterone and the superovulatory response in Murrah buffaloes pretreated with GnRH agonist.

Selection of experimental animals
 
The study was carried out on buffaloes (n=27) at University Instructional Dairy Farm. The buffaloes selected were aged between 4-8 years, clinically sound, normal cyclic, healthy genitalia with patent cervix during diestrus. All the buffaloes were kept under similar conditions of feeding and management practices.
 
Experimental groups
 
Animals were randomly divided into three groups; (n=9; each group) and subjected to superovulatory treatment as:
 
Group I (GnRH @ 10 µg)
 
GnRH (10 µg) intramuscular was given on day 7 (day 0 = day of estrus) of estrus before initiating superovulatory treatment. From day-10 buffaloes were injected with total 600mg follicle stimulating hormone (FSH; Folltropin®-V, Bioniche Animal Health Canada Inc.) in 10 divided tapering doses at 12 hr interval over 5 days, i.e. 80:80, 70:70, 60:60, 50:50, 40:40 mg, morning and evening by IM route. Luteolysis was induced by Cloprostenol Sodium (500 µg IM) given with seventh and eighth dose of Folltropin in order to induce super estrus 36-48h later.
 
Group II (GnRH @ 06 µg)
 
GnRH (06 µg) was administered intramuscular on day 7 before initiating superovulatory treatment. From day-10 onwards same treatment was administered as in group-I.
 
Group III (Control)
 
Superovulatory regime was started from day 10 as in group-I, without pretreatment with GnRH.
 
Estrus induction and detection of base heat
 
The buffaloes selected were either in natural or induced to estrus with 500 µg intramuscular injection of Cloprostenol Sodium in animals having mature corpus luteum. The estrus detection was done morning and evening by close observation for external signs, such as bellowing, mucus discharge from vulva, mounting, frequent micturition, swollen and edematous vulva and confirmed by rectal palpation. Treatment was started from day-7 onwards as stated in experimental groups.
 
Insemination of donors during superovulatory estrus
 
During the superovulatory estrus, the buffaloes were fixed timed artificially inseminated at 36 hr after the first prostaglandin injection for three times at 12 hr interval with frozen-thawed semen of one elite bull.
 
Evaluation of superovulatory response and embryo collection
 
The superovulated buffaloes were examined per-rectally on day of embryo collection to evaluate superovulatory response. Both the ovaries were palpated gently to count the number of corpus luteum (CL) and unovulatory follicles. Superovulatory response for each animal was recorded based on total number of CL present on both ovaries. The embryos were collected non-surgically after 132 hr (5.5 day) of first artificial insemination using 18 G Rusch catheter (75 cm length). Embryos were collected by flushing of uterus as described by Singhal et al., (2021). Briefly, about 500 ml of Dulbecco’s phosphate buffer saline (DPBS) containing 0.1% bovine serum albumin (BSA) was used for flushing of each uterine horn separately. Following the process of embryo flushing, 30 ml Gentamycin was infused into uterus to protect against any possible infection and Cloprostenol Sodium (500 µg) was administered for luteolysis to bring animal in estrus. After flushing, the media collected into the Emcon filter was transferred to petridish and searched thoroughly for embryos under stereozoom microscope at 20-40X magnification.
 
Hormone estimation
 
About 5 ml blood without anticoagulant was collected on different days of treatment in sterilized glass tube. Blood serum was separated, centrifuged at 3000 rpm for 15 minute and was transferred into sterilized serum vials. The samples were stored at -20°C till analysis. Progesterone hormone was estimated in serum samples by Radioimmunoassay (RIA) using RIA kits (Immunotech®, Bakmann coulter Company, France).
 
Statistical analysis
 
Data obtained during study was analysed for mean, standard error and coefficient of correlation. Difference between means were compared using students‘t’ test.

Progesterone profile and its correlation with superovulatory parameter
 
In our study, control and group II from day 7 to day 10 showed increase in P4 levels but in group I these values fluctuates (Table 1). On day of initiation of superovulatory regime P4 value ranged between 2.11 to 3.76 ng/ml with an overall value of 3.09 ng/ml was in concurrence with Ravi et al., (2011). The serum progesterone level at the initiation of superovulatory treatment (D-10) was positively but non-significantly correlated with the number of CL and embryo recovery in all three groups (Table 2). These findings are in agreement with several other studies reported earlier (Kharche et al., 2002; Heleil and Deeb, 2010). Similarly, Madan et al., (1988) claimed that buffaloes with >2ng/ml plasma P4 during pretreatment has better superovulatory response than those having <2 ng/ml P4. Moreover, blood progesterone level around 4-5 ng/ml provided by progesterone implant (CIDR) used during superovulation in buffalo, significantly improved the superovulatory response (Neglia et al., 2010). However, the present findings were contradictory with other workers (Taneja et al., 1995) who fail to establish any correlation between P4 concentration on the day of FSH treatment and superovulatory response. The variations in plasma progesterone concentration might due to different agroclimatic zones and weather conditions prevailing under these studies (Phogat et al., 2016). In our study, the increased serum progesterone concentration from day 10 to 13, might be due to the luteotropic action of administered gonadotropin (Jindal et al., 1988) or due to presence of functional CL.
 

 

 
After 12 hr of PGF2a treatment progesterone concentrations declined nearly to 1.5 ng/ml in all experimental groups and further reduced to less than 0.75 ng/ml within 24 hr in group I and II. Similarly, other preliminary studies indicated decline in progesterone levels below 1.0 ng/ml (Beg et al., 1997) or further reduced to lower than 0.5 ng /ml (Misra et al., 2000) within 24 h of PG treatment.
       
From day 17 to embryo flushing, there was continuous increase in progesterone levels in all three groups probably due to the secretion of progesterone by multiple CL or due to luteinization of large unovulated follicles (Ireland and Roche, 1982). The present study observed the overall serum progesterone levels of 11.15±0.81 ng/ml on day of embryo flushing, which was equivalent to earlier reported values (Ravi et al., 2011), but relatively higher (12.90±1.4 ng/ml) and lower (9.05 ng/ml) levels were observed by Halbert et al., (1989) and Dutta et al., (1992), respectively. Our work recorded, P4 levels on day 20 (day of embryo flushing) were significantly (P<0.05) positively correlated with the number of CL and recovered embryos for all three groups as were estimated by few other scientists (Misra et al., 2000) in Murrah buffaloes.

Thus, the present study concluded that, GnRH pretreatment improved the progesterone concentration and superovulatory response in terms of ovulation rate, CL formation and embryo recovery in Murrah (Bubalus bubalis) buffaloes.