Polymorphism in 5′ untranslated region of heat‐shock protein 70 gene as marker of post‐partum anoestrus in Murrah buffaloes

The enormous production potential of buffaloes has never been accomplished due to various reproductive insufficiencies. Among them, post‐partum anoestrus, a multifactorial disorder, is predominant but any genetic association is yet to be established. This study focused to identify novel polymorphisms in heat‐shock protein 70 (HSP70) gene and its possible association with post‐partum anoestrus in Murrah buffaloes. A 579‐bp fragment from 5′ untranslated region of HSP70 gene was amplified by polymerase chain reaction from blood genomic DNA of 614 animals maintained under similar management conditions. In phase‐I experiment, custom sequencing and restriction enzyme (RE) digestion of the amplified fragment were performed in 40 buffaloes with similar post‐partum oestrous conditions over previous consecutive three or more gestations—20 animals each showing post‐partum anoestrus (>120 days after parturition) and normal cyclicity (<65 days after parturition). While in phase‐II experiment, herd screening by RE analysis was performed in remaining 574 animals. Four transversions at T‐75G, C+31G, T+38G and C+97A and three transition mutations at T‐153C, T+33C and A+44G positions were observed. Polymorphism at T+38G site revealed significant (p < .05) variation, where homozygous G was present only in post‐partum anoestrous animals while nucleotide T was present randomly in both groups of phase‐I animals whereas phase‐II experiments revealed homozygous G in 55 animals. Regression analysis in relation to average post‐partum interval against genotypic frequencies at T+38G also depicted significant association. HSP70 gene polymorphism at T+38G position can therefore be used as genetic marker for excluding probable post‐partum anoestrous buffaloes from herd for breeding programmes.     

Relationship of temperament with oestrous behaviour,resumption of ovarian cyclicity and milk yield in post‐partum Murrah buffaloes

This study was designed to investigate the relationship of temperament with oestrus, resumption of ovarian cyclicity and milk yield in post‐partum buffaloes. For this study, 102 post‐partum Murrah buffaloes were observed for temperament in open paddock and during milking. Based on the temperament score (1–5 score), they were classified into docile, slightly restless, restless, aggressive and nervous. Oestrous behaviour was monitored twice daily in all the buffaloes along with milk yield (weekly), body weight (BW) and body condition score (BCS) at day 0, 30, 60 and 90 post‐partum. Based on the temperament score, the number of buffaloes classified as docile, slightly restless, restless and aggressive was 37 (36.27%), 28 (27.45%), 30 (29.41%) and 7 (6.86%), respectively, but none under nervous category. Number of buffaloes resuming cyclicity in various temperaments groups were as follows: docile (n = 35; 94.59%), slightly restless (n = 22; 78.57%), restless (n = 20; 66.67%) and aggressive (n = 7; 85.71%). Temperament was not correlated (p = .128; r=?.152) with oestrous behaviour. But, temperament was correlated (p < .0001; r = .384) with the resumption of cyclicity as docile and slightly restless buffaloes resumed cyclicity (p < .0016) earlier. Temperament of buffaloes was weakly correlated (p = .0017; r=?.306) with milk production with higher (p < .0358) total milk yield in docile (1022 ± 23.75 kg) and slightly restless buffaloes (948 ± 35.86 kg) as compared to other temperament groups. In summary, temperament of post‐partum buffaloes was related with resumption of ovarian cyclicity and milk yield, but not oestrous behaviour.     

Reproductive Patterns in the Non‐Breeding Season in Asinina de Miranda Jennies

This study aims to characterize the reproductive patterns in Asinina de Miranda jennies during the non‐breeding season. Reproductive activity was surveyed in 12 females, aged between 3 and 18 years old, using ultrasound and teasing with a jack. The animals were monitored from September to April, six in each consecutive year. Of these 12 females, nine showed disruption to the normal pattern of ovarian activity during the non‐breeding season. Loss of normal cyclicity included anoestrus (41.7%), silent ovulatory oestrus (25%), and persistence of corpus luteum (8.3%). Only three females maintained a regular cyclic pattern with oestrous behaviour during the non‐breeding season. Anoestrus began in early November and lasted for an average of 147 ± 28 days (113–191 days), ending near to the spring equinox. Onset of silent oestrous cycles began more erratically, between October and February. In both groups the first behavioural ovulation of the year occurred around the time of the spring equinox. Disrupted reproductive activity was preceded by a shorter oestrous cycle only in females entering anoestrus. The mean follicle size in the first ovulation of the year was larger than in the reproductive season (44.7 ± 2.45 mm vs 39.2 ± 3.60 mm) in anoestrous jennies with protracted oestrus. Though age and body condition score (BCS) were associated, changes in BCS below a threshold of four points (for anoestrus) and five points (for silent oestrus) contributed greatly to disruption of reproductive cycles. BCS in females with regular oestrous cycles during the winter season remained unchanged or exceeded five points prior to the winter solstice.     

Clinical efficacy of intrauterine cephapirin benzathine administration on clearance of uterine bacteria and subclinical endometritis in postpartum buffaloes

This study examined the effect of single IU administration of cephapirin on clinical recovery, clearance of uterine bacteria and reproductive performance of postpartum buffaloes with subclinical endometritis (SCE). Buffaloes (n = 86) at 35 days postpartum (DPP) with >10% polymorphonuclear (PMN) cells in endometrial cytosmears were designated as positive (SCEP, n = 29), and buffaloes with ≤10% PMN cell were designated as negative (SCEN, n = 57) for SCE. Out of 29 positive buffaloes, 15 were administered a single intrauterine dose of cephapirin benzathine on 40 DPP (SCEP‐CB), while the remaining 14 animals were kept as untreated control (SCEP‐C). All animals were observed regularly for oestrous signs and were again subjected to cytobrush sampling on the first postpartum (FPP) oestrus. Buffaloes positive for SCE at 35 DPP were later considered “recovered” if their PMN cells dropped to ≤5% on the FPP oestrus. Presence of Escherichia coli, Arcanobacterium pyogenes and Fusobacterium necrophorum in uterus was detected based upon PCR amplification of genes related to bacteria‐specific virulence factors. A total of 66.7% of SCEP‐CB group buffaloes recovered as compared to 28.6% in SCEP‐C (χ² = 4.21; p < 0.05). Rate of bacterial clearance did not differ between treated (38.5%) and untreated buffaloes (8.3%) (χ² = 1.67; p > 0.05). The median days to first service did not differ significantly (p > 0.05) among the three groups, whereas cephapirin administration reduced (p < 0.05) the days open by 14 days in SCEP‐CB compared to SCEP‐C buffaloes. SCEP‐CB buffaloes were as likely to conceive as SCEN, whereas SCEP‐C had 0.28 hazard ratio for pregnancy. In conclusion, a single treatment with cephapirin benzathine at 40 DPP improved the reproductive performance of buffaloes with subclinical endometritis.     

Follicular dynamics and colour Doppler vascularity evaluations of follicles and corpus luteum in relation to plasma progesterone during the oestrous cycle of Surti buffaloes

With an objective to evaluate the follicular dynamics and vascularity changes in follicles and corpus luteum, the ovaries of cyclic Surti buffaloes (n = 9) were examined daily sequentially by transrectal B‐mode and colour flow mode (CFM) ultrasonography starting from the day of oestrus till the onset of next oestrus. Higher proportion of buffaloes evidenced one‐wave cycle (66.66%) compared to two‐wave cycle (33.34%) with none showing a three‐wave cycle. The dominant follicle of the first follicular wave was the ovulatory follicle and persisted for 19.70 ± 0.50 days compared to its persistence for 16.5 ± 1.45 days in a two‐wave cycle. The maximum diameter of the ovulatory follicle in a one‐wave and two‐wave cycle did not differ yet their linear growth rates were significantly lower (p < 0.01) in a one‐wave cycle. Colour flow mode examination of follicles revealed that the percentage of follicles with detectable blood flow in the subsequently determined largest follicle (dominant follicle) was not different from that in the second largest follicle before follicle deviation. The blood flow in the dominant follicle increased significantly on the day of oestrus. The mean diameter and blood flow to the corpus luteum (CL) increased linearly and significantly from Day 5 of oestrus till Day 13 after which both parameters started declining. At or around Day 16, there was precipitous fall in the blood supply to the CL and CL diameter that continued declining thereafter to reach the lowest around Day 20 of the oestrous cycle. Rise in plasma progesterone concentrations was synchronous to CL diameter and vascularity and showed significant and positive correlations. It was concluded that Surti buffaloes evidence a preponderance of one‐wave follicular growth pattern with a significant increase in the vascularity of ovulatory follicle on the day of oestrus and corpus luteum on Day 13 of the oestrous cycle.     

Vaginal Histological Changes after Using Intravaginal Sponges for Oestrous Synchronization in Anoestrous Ewes

To characterize the histological and cytological vaginal changes generated by the use of intravaginal sponge (IS) applied in oestrous synchronization treatments in ewes during mid‐non‐breeding season. Thirty‐five multiparous ewes were allocated to three experimental groups according to the moment in which the samples were taken: (i) ewes treated with IS containing 60 mg of medroxyprogesterone acetate for 14 days, sampled the day of IS removal (group ISR; n = 10), (ii) or after sponge removal at time of oestrus or 72 h after removal (group AR; n = 14) and (iii) ewes without sponge treatment that were sampled at the day of IS removal of the other groups (group CG; n = 11). Vaginal biopsies and cytological samples were taken from the anterior vaginal fornix area. The vagina of the CG group had a stratified squamous epithelium with a moderate degree of cellular infiltration with lymphocytes and plasma cells in the lamina propia. Treated ewes (ISR and AR) had epithelial hyperplasia and hypertrophy. ISR ewes had haemorrhage and perivascular infiltrate and an increased number of epithelial cells, neutrophils, macrophages and erythrocytes at IS removal. The use of IS generated histological and cytological alterations in the vaginal wall when used for oestrous synchronization in anoestrous ewes.     

Induction of Ovulation of Ovulatory Size Non‐Ovulatory Follicles and Initiation of Ovarian Cyclicity in Summer Anoestrous Buffalo Heifers (Bubalus bubalis) Using Melatonin Implants

This study was conducted on summer anoestrous buffalo heifers to monitor the efficacy of melatonin for induction of ovulation and ovarian cyclicity. During pre‐treatment period of 24 days, the ovarian dynamics of five cycling and 10 summer anoestrous heifers was monitored on each alternate day using a transrectal ultrasound scanner. Thereafter, during treatment period, these 10 anoestrous heifers along with additional seven anoestrous heifers were randomly allocated into non‐implanted (n = 5) and implanted (n = 12, one melatonin implant/50 kg, 18 mg melatonin/implant) group. Non‐implanted heifers were monitored on each alternate day till the confirmation of second‐ovulation in implanted heifers. Pre‐treatment period revealed the presence of dominant follicles in anoestrous heifers which attained the diameter comparable with ovulatory follicles of cycling heifers but failed to ovulate and regressed. Between 6 and 36 days (15.3 ± 2.9 days) post‐treatment, all the implanted heifers (p < 0.05) exhibited ovulation of dominant follicles; however none of the non‐implanted heifers ovulated during the corresponding period. The first‐interovulatory period in implanted heifers ranged between 8 and 28 days (18.0 ± 1.8 days). The implanted heifers with short (≤16 days) interovulatory period had short‐lived corpus luteum (CL) that had smaller diameter and secreted less progesterone (p < 0.05). The diameter of CL was large (p < 0.05) and plasma progesterone was high (p < 0.05) following second‐ovulation compared with first‐ovulation in implanted heifers. In conclusion, using melatonin implants, ovulatory size nonovulatory follicles observed in summer anoestrous buffalo heifers can be successfully ovulated to initiate ovarian cyclicity.     

Interrelationship between milk constituents,serum oestradiol and vaginal mucus indicators of oestrus in Egyptian buffaloes

The intensity of heat signs in buffaloes is generally low and the incidence of suboestrus varied from 15 to 73% (Buffalopedia). The objective of this study was to investigate the feasibility of monitoring the changes in some milk constituents, oestradiol levels and electrical conductivity of vaginal mucus during peri‐oestrous period in prediction of the timing of oestrus in buffaloes. Twenty‐one Egyptian buffaloes aged 3–9 year, 1st–6th lactations, were examined by oestrous detector and ultrasonographically for monitoring the ovarian and uterine activity for 7 days around the time of standing oestrus. Sodium, potassium, chloride and lactose were assayed in aqueous phase of milk; besides, oestradiol was estimated in serum. Current results declared highly significant acute changes in milk constituents at the time of oestrus characterized by peaking of chloride and sodium levels and lowering of potassium and lactose values. The alternation in milk composition when arranged in decreasing order of magnitude, sodium was the highest (77.78 ± 0.69%), followed by chloride (61.60 ± 1.52%) and potassium (?58.14 ± 10.89%). Concomitantly, milk lactose decreased by 26.07 ± 7.97% compared to baseline levels. Synchronously, vaginal electrical resistance (VER) showed a significant (p < 0.01) decrease, but serum oestradiol 17β levels surged (59.93 ± 7.29 pg/ml) on day of oestrus. Serum oestradiol level was negatively correlated with VER (r = ?0.577), potassium (r = ?0.661), positively correlated with chloride (r = 0.707) and sodium (r = 0.579) and not correlated with lactose levels. These results for the first time suggested that the changes in constituents of milk during peri‐oestrous period may be used as a practical non‐invasive indicator for oestrous detection and prediction of ovulation in Egyptian buffaloes.     

Milk progesterone enzyme-linked immunosorbent assay as a tool to investigate ovarian cyclicity of water buffaloes in relation to body condition score and milk production

Background

Application of assisted reproductive technologies in buffaloes is limited to some extent by farmers’ inability to detect oestrus because of its poor expression. The present study aimed at investigating reliability of a milk progesterone enzyme-linked immunosorbent assay (ELISA) to assess the ovarian cyclicity during post partum, oestrus and post-breeding periods in water buffaloes.

Methods

Progesterone concentrations were measured by an ELISA in milk of 23 postpartum buffaloes in relation to oestrus, pregnancy, body condition score (BCS) and milk production. Two milk samples were taken at 10 days intervals, every month starting from day 30 and continued to day 150 post partum. BCS and milk production were recorded during sample collection. Milk samples from bred buffaloes were collected at Day 0 (day of breeding), Days 10–12 and Days 22–24. Defatted milk was preserved at −80°C until analysis. Pregnancy was confirmed by palpation per rectum on Days 70–90.

Results

Seventeen buffaloes had 47 ovulatory cycles, one to four in each, 13 were detected in oestrus once (28 % oestrus detection rate). Progesterone concentration ≥1 ng/ml in one of the two 10-day-interval milk samples reflected ovulation and corpus luteum formation. The intervals between calving to first luteal activity and to first detected oestrus varied from 41 to 123 days (n = 17) and 83 to 135 (n = 13) days, respectively. Eight buffaloes were bred in the course of the study and seven were found pregnant. These buffaloes had a progesterone profile of low (<1 ng/ml), high (≥ 1 ng/ml) and high (≥ 1 ng/ml) on Day 0, Days 10–12 and Days 22–24, respectively. Buffaloes cycling later in the postpartum period had fewer missed oestruses (P < 0.05). Buffaloes with a superior BCS had a shorter calving to oestrus interval and produced more milk (P < 0.05).

Conclusions

Milk progesterone ELISA is a reliable tool for monitoring ovarian cyclicity and good BCS may be an indicator of resuming cyclicity in water buffalo.     

Comparative efficacy of oestrus synchronization protocols in buffalo (<Emphasis Type="Italic">Bubalus bubalis</Emphasis>)

This study was conducted to test the efficacy of gonadotropic hormone (GnRH)-based synchronization protocols (Ovsynch, Heatsynch, and Ovsynch Plus) in buffaloes under field condition. Based on anamnesis and transrectal palpation twice at 10-day interval and serum progesterone (P₄) concentration, 150 anoestrous buffaloes and delayed pubertal heifers were selected to induce oestrus using GnRH-based protocols. These selected animals were randomly divided into three groups: group I: Ovsynch (n = 50), group II: Heatsynch (n = 50), and group III: Ovsynch Plus (n = 50) regimen. Before treatment initiation, blood samples were collected for P₄, beta-hydroxy butyric acid (β-OHB), and mineral estimation, in addition to the monitoring of oestrus signs. In this investigation, no significant difference (P > 0.05) in oestrus signs was deduced among three groups. Oestrus induction rate (OIR) was comparable (P > 0.05) among the groups (Ovsynch 82%, Heatsynch 86%, and Ovsynch Plus 88%). Conception rate (CR) following fixed time artificial insemination (FTAI) was slightly higher with Ovsynch Plus group (28%) as compared to Ovsynch (24%) and Heatsynch (18%) groups, though non-significant. Furthermore, serum glucose, β-OHB, macrominerals (calcium, potassium, and magnesium), and trace minerals (copper, zinc, and iron) remained comparable (P > 0.05) among the groups. In conclusion, all the protocols (Ovsynch, Heatsynch, and Ovsynch Plus) are efficient in oestrus induction in anoestrous buffaloes under field condition with Ovsynch Plus protocol resulting in higher CR as compared to other protocols.     

Validation of Bovine Oestrous‐Specific Synthetic Molecules with Trained Scent Dogs; Similarities Between Natural and Synthetic Oestrous Smell

Oestrous detection is crucial for successful dairy cow reproduction. Bulls identify cows in oestrus by oestrous‐specific odours especially in urine and vaginal fluid. These have been used to train dogs to detect cows in heat. To improve and simplify the dog training, a spray containing synthetic oestrous molecules was developed. The objective of this study was to test the spray on similarities to the natural substance thus to assess its suitability as a training substance for heat detection dogs. Ten privately owned dogs of various breeds were trained. Dogs should be trained either to differentiate natural vaginal fluid from cows in oestrus and dioestrus (n = 5), or spray with or without synthetic oestrous molecules (n = 5). Dogs trained on natural fluid and on spray could detect the oestrous odour they had been trained on with an overall accuracy of 69.0% and 82.4%, respectively (p = 0.019). To validate the synthetic molecules, dogs trained with synthetic molecules had to detect oestrous odour in natural fluid without further training (accuracy 37.6%). Dogs trained on natural fluid detected the synthetic molecules with an accuracy of 50.0% (50% vs 37.4%, p < 0.05). Dogs can recognize natural vaginal fluid from cows in oestrus after they have been trained with synthetic oestrous molecules, but accuracy needs to be improved.     

Comparison of Different Treatments for Oestrous Induction in Seasonally Anovulatory Mares

The aim of this study was to evaluate the effects of different treatments for induction and synchronization of oestrus and ovulation in seasonally anovulatory mares. Fifteen mares formed the control group (C), while 26 mares were randomly assigned to three treatment groups. Group T1 (n = 11) were treated with oral altrenogest (0.044 mg/kg; Regumate^®) during 11 days. Group T2 (n = 7) was intravaginally treated with 1.38 g of progesterone (CIDR^®) for 11 days. In group T3 (n = 8), mares were also treated with CIDR^®, but only for 8 days. All mares received PGF2α 1 day after finishing the treatment. Sonographic evaluation of follicles, pre‐ovulatory follicle size and ovulation time was recorded. Progesterone and leptin levels were analysed. Results show that pre‐ovulatory follicles were developed after the treatment in 88.5% of mares. However, the pre‐ovulatory follicle growth was dispersal, and sometimes it was detected when treatment was not finished. While in mares treated with intravaginal device, the follicle was soon detected (1.5 ± 1.2 days and 2.3 ± 2.0 days in T2 and T3 groups, respectively), in T1 group, the pre‐ovulatory follicle was detected slightly later (3.9 ± 1.6 days). The interval from the end of treatment to ovulation did not show significant differences between groups (T1 = 13.1 ± 2.5 days; T2 = 11.0 ± 3.6 days; T3 = 13.8 ± 4.3 days). The pregnancy rate was 47.4%, similar to the rate observed in group C (46.7%; p > 0.05). Initial leptin concentrations were significantly higher in mares, which restart their ovarian activity after treatments, suggesting a role in the reproduction mechanisms in mares. It could be concluded that the used treatments may be effective for oestrous induction in mares during the late phase of the seasonally anovulatory period. Furthermore, they cannot synchronize oestrus, and then, it is necessary to know the reproductive status of mares when these treatments are used for oestrous synchronization.     

Effect of exogenous administration of oxytocin on postpartum follicular dynamics,oestrous rate and ovulation in Nili-Ravi buffaloes

Based on different surveys, dairy farmers are concerned about extensive use of exogenous oxytocin in buffaloes, which is being held responsible for reproductive problems including irregular oestrous cycle and delayed ovulation. For these concerns, effects of oxytocin injection on postpartum follicular dynamics, postpartum oestrous interval (PEI), oestrous length, the interval from onset of estrus to ovulation and blood progesterone (P4) were studied in Nili-Ravi buffaloes. For this purpose, 23 animals within 1 week after calving were randomly divided into three groups: without oxytocin (CON; n = 7), 10 i.u. oxytocin (LOW; n = 8), 30 i.u. oxytocin – (HIGH; n = 8) and used to record the PEI for the study period of 154 days. At subsequent estrus, three buffaloes from each group (not served) were selected randomly to monitor two cycles for 6 weeks. Transrectal ultrasonography was performed to evaluate follicular and corpus luteum (CL) development, and blood sampling was done for progesterone (P4) analysis. These results revealed that postpartum oestrous interval (PEI) decreased significantly in oxytocin-treated groups. The number of small, medium and total follicles on the left ovary was significantly higher in the HIGH group. However, an overall number of small and total follicles on both right and left ovaries was significantly higher in CON and HIGH groups. On the other hand, there was no difference in the number of follicles on the right ovary among all treatment groups. The same was true for the size of pre-ovulatory follicles, CL, P4 concentrations and oestrous cycle length. The intervals from onset of estrus to ovulation and from standing estrus to ovulation were increased considerably in the HIGH group. It is concluded that exogenous oxytocin administration resulted in the shortening of PEI but triggered a delay in ovulation. Moreover, a higher dose of oxytocin could stimulate the growth of small, medium, and total follicles in postpartum Nili-Ravi buffaloes.     

Metabolic blood profile of beef heifers during oestrous and non‐oestrous states

Haematological metabolic profiles in heifers could contribute to the development of proxies for oestrous detection and provide clues to further characterize biological changes during oestrus. One hundred and seven beef heifers were observed for oestrous behaviour twice daily for 124 days. Feed intake and productive performance (body weight and composition) traits were measured, and feed efficiency was determined using residual feed intake (kg DM/day). Blood plasma samples were collected when signs of oestrus were observed and every 30 ± 2 days. Heifers were considered in oestrus (n = 71) when plasma progesterone concentrations were <0.6 ng/ml. Least square means of blood metabolic parameters were compared between oestrous and non‐oestrous states and within oestrous groups according to performance traits and age. Heifers in oestrus exhibited higher concentrations of alkaline phosphatase, aspartate aminotransferase (AST), beta‐hydroxybutyric acid, creatine kinase (CK) and triiodothyronine (T3) than heifers in non‐oestrus. Heifers in oestrus revealed lower osmolality and concentrations of calcium, sodium and total protein than during non‐oestrus. Younger (and smaller) heifers had greater concentrations of CK, gamma‐glutamyl transferase (GGT), glucose and sodium than the older heifers. Heifers with lower fatness had increased osmolality and concentrations of cholesterol, CK, phosphorus, sodium and reduced T3 levels. Feed efficient heifers had greater levels of AST, cholesterol and GGT than inefficient heifers. Blood plasma parameters may be complementary to oestrous detection upon further validation; effects of age, feed efficiency, body size and body composition should be considered to optimize this haematological assessment.     

Influence of Gonadotrophin‐Induced First Oestrus on Gilt Fertility

The aim of this study was to determine the association between the oestrous response of pre‐pubertal gilts to gonadotrophin injection or boar exposure and their subsequent farrowing rate and litter size. At 154 days of age, randomly selected pre‐pubertal gilts received an intramuscular injection of 400 IU equine chorionic gonadotrophin plus 200 IU human chorionic gonadotrophin (PG600^®; Merck Animal Health; n = 181). From the remaining pool of animals not treated with hormones, the first gilts showing signs of oestrus were selected to act as controls (n = 201). Boar exposure began at 155 days of age for both groups, and gilts were bred at a weight of approximately 130 kg. Comparisons were made between PG600^®‐treated gilts exhibiting oestrus or not within 7 days post‐injection (early and late responders, respectively) and control gilts exhibiting oestrus or not within 30 days after beginning of boar exposure (select and non‐select control gilts, respectively). By 162 days, oestrus was detected in 67.5% of PG600^®‐treated gilts compared with 5.7% of control gilts (p < 0.0001). The proportion of animals observed in oestrus at least three times before breeding was greater for select control gilts compared with early and late responder PG600^®‐treated gilts (p ≤ 0.001). There were no significant differences in farrowing rate and litter size between the four treatment groups. These data indicate that PG600^® is an effective tool to induce an earlier oestrus in gilts, that subsequent farrowing rate and born alive litter size compare favourably to that of select gilts and that gilts failing to respond promptly to hormonal stimulation do not exhibit compromised fertility.     

Evaporative cooling in late gestation heat‐stressed Murrah buffaloes increases efficiency of next reproductive cycle

Evaporative cooling during late gestation period improves post‐partum reproductive performance in Murrah buffaloes. To prove this hypothesis, sixteen pregnant dry Murrah buffaloes at sixty days pre‐partum were selected and divided into two groups of eight animals each. Group 1 of buffaloes (Cooled/CL) was managed under fan and mist cooling during dry period, whereas second group of buffaloes (non‐cooled/NCL) remained without the provision of cooling. After parturition, all the animals were managed under evaporative cooling till the end of experimental period. Reproductive performance in cooled (CL) and non‐cooled (NCL) groups, respectively, viz. 1st and 2nd ovulation from calving (48.63 ± 2.41, 69.25 ± 2.34 days and 57.75 ± 3.35, 93.63 ± 2.84 days); calving to conception interval (117.88 ± 4.21 days and 117.88± 4.21 days); conception rate (87.5% ± 2.16% and 57% ± 2.26%); and follicular diameter at the time of 1st and 2nd ovulation (14.84 ± 0.16, 15.75 ± 0.13 mm and 12.65 ± 0.13, 13.35 ± 0.11 mm) varied significantly (p < .05). Total peak oestrogen concentration was significantly (p < .05) higher in cooled (26.7 ± 1.32 pg/ml) relative to non‐cooled (20.7 ± 1.22 pg/ml) buffaloes. Time from onset of oestrus to ovulation varied significantly (p < .05) in cooled (32 ± 2.22 hr) and non‐cooled (40 ± 2.86 hr) buffaloes. The peak progesterone concentration reached to (4.25 ng/ml) in cooled group and (4.16 ng/ml) in non‐cooled group after first ovulation.     

Induction of oestrus by administering Inhibin antiserum along with equine chorionic gonadotropin in anoestrous bitches

As dogs experience oestrus only once or twice a year, it is necessary to establish an effective method of oestrous induction for efficient breeding. In the present study, we evaluated inhibin antiserum (IAS) on oestrous induction in anoestrous females. Bitches were administered 0.5 ml/kg IAS or a mixture of 50 IU/kg equine chorionic gonadotropin (eCG) and 0.5 ml/kg IAS and 500 IU human chorionic gonadotropin (hCG) administered 7 days after the mixture injection. As a control, bitches received 50 IU/kg eCG, with 500 IU hCG administered 7 days after eCG injection. Blood-tinged vaginal discharge, vulvar swelling, plasma progesterone concentrations and ovarian follicular development were assessed from day 0 to day 14. IAS alone injection did not induce oestrus in bitches at the anoestrous stage. Conversely, vulvar swelling, blood-tinged vaginal discharge and an estimated luteinizing hormone (LH) surge appeared on days 3–7, days 3–6 and days 7–9 after the IAS+eCG mixture injection, respectively, in all five bitches at the anoestrous stage. The average number of developing and ovulated follicles in bitches administered IAS+eCG was 8.8 and 9.6 respectively. A single eCG injection followed by hCG induced oestrous signs, with an average of 8.3 developing follicles and 4.5 ovulated follicles. This study revealed that IAS alone did not induce oestrus, but when IAS was used in combination with eCG, it induced oestrus and promoted a considerable number of ovulations in anoestrous dogs.     

Higher doses of Melatonin affect ovarian and middle uterine arteries vascular blood flow and induce oestrus earlier in acyclic ewes

The present study aimed to evaluate the effect of a single intramuscular injection of melatonin on response of oestrus, rate of conception, ovarian and uterine blood supply during the non-breeding season. Melatonin powder was dissolved in dimethyl sulfoxide (DMSO) and olive oil and injected in two dose of 25 mg (MEL-1; n = 10) and 50 mg (MEL-2; n = 10) in May. Ten ewes were served as the control (CON). Results showed that oestrous response was higher in the MEL-2 group than in other groups (p < .01). The time to onset of oestrus in both the MEL-1 and MEL-2 groups was significantly shorter, compared with the CON group (p < .01). Pregnancy rates reached 80% and 90% at 45 days of gestation. Doppler indices of the right and left ovarian artery were lower in the melatonin groups than in normal groups (p < .01). Both Doppler indices in uterine arteries were not different among two treatment groups (p > .05), but there was a significant difference when compared with the CON group (p < .01), no difference found between two treatment group in the Doppler velocities of middle uterine arteries (p > .05). In conclusion, the definite changes in the studied parameters showed two novel findings; (a) the increase in melatonin dose results in increasing the blood flow expressed by increased Doppler velocities in fatty tailed ewes, (b) as well as, melatonin can initiate the breeding season regardless of the season earlier by 1–2 months in ewes.     

Nasal immunization with inhibin DNA vaccine delivered by attenuated Salmonella choleraesuis for improving ovarian responses and fertility in cross‐bred buffaloes

This study was conducted to determine the effect of immunization with inhibin DNA vaccine delivered by attenuated Salmonella choleraesuis on ovarian responses and fertility in cross‐bred buffaloes. A total of 134 cross‐bred buffaloes were divided into four groups: groups T1 (n = 34), T2 (n = 35) and T3 (n = 31) were nasal immunized twice a day with 10 ml of 1 × 10¹⁰ CFU/ml of the C501 (pVAX‐asd‐IS) vaccine for 5, 3 and 1 day, respectively. Group C (n = 34) was nasal immunized with 10 ml PBS for 5 days. All animals were immunized twice with an interval of 14 days and administered with 200 μg of a GnRH analogue on day 28, 0.5 mg PGF_2α on day 35 and 200 μg of the same GnRH analogue on day 37. TAI was performed at 18 and 24 hr after the second GnRH treatment. Fourteen days after primary immunization, C501 (pVAX‐asd‐IS) elicited significant immune responses, and anti‐inhibin IgG antibody titres in group T1 were significantly higher (p < .01) than groups T3 and C. After the second GnRH treatment, the growth speed of the dominant follicles in group T1 was significantly faster (p < .05) than groups T3 and C. The number and diameter of large follicles (≥10 mm) as well as ovulatory follicles in group T1 were the greatest in all groups, resulting in a greater conception rate in buffaloes with positive anti‐inhibin antibodies. These results demonstrate that immunization with the C501 (pVAX‐asd‐IS) vaccine, coupled with the Ovsynch protocol, could be used as an alternative approach to improve reproductive performance in cross‐bred buffaloes.     

Urine levels of luteinizing hormone as predictor of the period of ovulation for advantage of timed‐artificial insemination in murrah buffalo (Bubalus bubalis)

Assessment of urine levels of luteinizing hormone (LH) for predicting the reproductive status of animals is in practice. The aim of this study was to predict the period of ovulation based on the urine levels of LH for timed‐artificial insemination to increase the conception rate in buffaloes, which are naturally silent‐oestrous animals. Level of LH in urine was assessed using ELISA, and a cut‐off LH concentration for prediction of ovulation period was obtained using receiver operating characteristic analysis. Artificial insemination was performed before‐ and after ‐positive prediction of ovulation period adopting this method, and the rates of conception were assessed. Urine LH level of 105 mIU/ml (n = 14) was derived as a cut‐off concentration which predicts the ovulation period. The buffaloes in the positively predicted group (day 1 or 2) inseminated via intracervical route had an increase in the conception rate (83.33%); however, the insemination in the before‐positive‐prediction group resulted in poor conception rates (day 0; 16.66%) compared to that of the naturally inseminated group (day 0; 75.0%). In conclusion, the urinary LH would possibly be a fairly reliable predictor of the ovulation period. The day when cut‐off LH concentration is obtained may be taken as the most favourable time for artificial insemination, so as to attain a much better rate of conception in the buffalo.