Estrus In Buffaloes Research Articles

Mucin and salt combination simulate typical fern-like pattern of buffalo saliva smear at estrus.

Estrus detection in buffaloes primarily relies on behavioral and physiological signs. Especially during summer, these signs are less prominent to recognize. Thus, estrus detection is a pronounced challenge within the realm of buffalo husbandry, particularly in the summer. Therefore, a simple and accurate estrus detection method is required for buffalo farmers. The observation of fern-like salivary crystallization patterns is one such simple method to detect estrus in buffaloes, bactrian camels, beagle bitches, and cows. However, the exact mechanism for the formation of typical fern-like is not known. We hypothesized that it might be because of the estrus-specific mucins and salts. To test this hypothesis, we prepared the smears by combining different concentrations of mucin type -2 (MUC2) and -3 (MUC3) with sodium chloride (NaCl). Microscopic examination confirmed that fern-like patterns resulted from a combination of the MUC3 and NaCl produced more realistic fern patterns than that of MUC2 or BSA with salt. To predict possible mucin and salt concentration showing natural fern-like patterns at the estrus stage in buffalo saliva, we constructed a guide tree of artificially generated fern-like patterns using an image analysis online tool. This computation analysis revealed that most of the natural buffalo estrus saliva samples showing typical fern-like patterns clustered in the cluster 2 of the guide tree comprising of 13 clusters. In the cluster 2, MUC3 in combination with the salt concentrations of 100, 150, and 250 mM was commonly found in a close proximity to the natural typical fern-like patterns of saliva smear of buffaloes at estrus. Conclusively, the buffalo saliva at estrus is predicted to have a gel-forming heavily glycosylated protein such as mucin along with at least 100 mM of NaCl. RESEARCH HIGHLIGHTS: Glycoprotein and salts combination replicates fern-like pattern of buffalo saliva at estrus. MUC3 and NaCl salt combination produces more realistic fern-like patterns compared with MUC2 or BSA and salt combination. MUC3 with NaCl at 100, 150, and 250 mM consistently resembled natural estrus saliva fern-like patterns. During estrus, buffalo saliva is expected to contain heavily glycosylated mucin and at least of 100 mM NaCl.

Estrus and ovulation synchrony of buffaloes (Bubalus bubalis): A review

Synchronizing estrus and ovulation in buffalo using timed inseminations (TAI) protocols circumvents the limitations associated with estrus detection. Spontaneous estrus in buffalo may achieve more conception rate on insemination than those subjected to synchronization of estrus and timed insemination (TAI). The advantages of insemination following synchronization programs include higher detection rates combined with decreased embryonic losses and reduction in days open. A synchonization programme should ensure control of estrus cycle which entirely depends on ovarian status of animals before starting any protocol. Ovarian follicular status at the beginning of treatment protocols plays vital role for effective response shown by animals. Studies show that more precise alterations in follicular waves may be needed to achieve better synchrony of ovulation and more fertility. The protocols which use Gonadotropic Releasing Hormone (GnRH), follicle stimulating hormone (FSH), luteinizing hormone (LH), equine chorionic gonadotropin (eCG), human chorionic gonadotropin (hCG), prostaglandins, progesterone and estradiol have been helpful in achieving the estrus and ovulation synchrony. Use of estradiol esters and progesterone per-vaginum implants have also been incorporated in synchronization programs. Synchronous emergence of follicular waves using trans-vaginal ultrasound guided follicular ablation and estradiol administration is a recent concept for synchronization in buffalo. Further, a recent approach has been to allow the follicle of second follicular wave to ovulate as the fertility has been lesser when first wave follicle ovulates. Moreover, controlling environmental influences, nutritional and managemental strategies remain pre-requisite to achieve desired goals though hormonal administration. This is more particular in buffaloes suffering from anestrum more so in low or non-breeding season and pre-pubetal anestrum. This review will cover the available documented literature regarding estrous cycle physiology with emphasis on follicular wave dynamics and the future area of work to be focused to achieve more reproductive efficiency in buffalo.

A hormone-based therapeutic strategy to reduce non-productive period in anestrus buffaloes during breeding and non-breeding season under field conditions

Sixty anestrus Murrah buffaloes were evaluated for the success rate of progesterone (P4) + estrogen (E2) + equine chorionic gonadotropin (eCG) protocol (Breeding season, BS=15; Non-breeding season, NBS=15) and estradoublesynch protocol (BS=15; NBS=15). The buffaloes subjected to P4+E2+eCG had higher (p>0.05) estrus induction response compared to estradoublesynch during BS (86.0 vs 80.0%, respectively) and NBS (73.3 vs 66.6%, respectively). Estrus period score at induced estrus in buffaloes subjected to either of protocols in BS or NBS was not different (p>0.05), however, pregnancy rate was lower (p<0.05) in buffaloes exhibiting <50 estrus period score in comparison to those exhibiting >50 score. Further, pregnancy rate (induced + spontaneous estrus) was better (p>0.05) using P4+E2+eCG compared to estradoublesynch protocol (73.3 vs 63.3%). In addition, the interval between start of a protocol and conception in P4+E2+eCG was less compared to estradoublesynch (p>0.05; 13.9±1.3 vs 17.5±3.1 days). In summary, P4+E2+eCG protocol is a better hormone-based strategy compared to estradoublesynch for anestrus buffaloes during breeding and non-breeding season under field conditions.

PGF<sub>2α</sub> induced estrus synchronization of native buffaloes in Bangladesh

Buffaloes have low fertility due to silent estrus, seasonal anestrous, delayed puberty and long calving interval. This study was aimed to investigate the effect of single and double doses of prostaglandin F2alpha (PGF2a) on synchronization of estrus in buffaloes. A total of 66 buffaloes were selected and subjected to estrus synchronization experiments: single dose PGF2a, and double dose PGF2a. In single dose PGF2a experiment, buffaloes were injected with either 500 or 875 µg of cloprostenol (PGF2a) and all buffaloes were examined to identify corpus luteum through rectal palpation before administration of PGF2a. In double dose experiment, buffaloes were injected with either 500 or 875 µg of cloprostenol (PGF2a) on Day 0 and Day 12 without identifying corpus luteum. The buffaloes were examined for estrus by collecting cervical mucus using universal artificial insemination gun from the mid cervix and timed artificial inseminations (TAI) were done after 72 hrs of last dose of PGF2α injection. The results showed that there were no significant differences in estrus rate (75±3.96% and 85±0.79%) single dose of PGF2α with 500 and 875 µg of cloprostenol, respectively. However, 875 µg of cloprostenol significantly (P<0.05) increased estrus rate in buffaloes than double doses with 500 and 875 µg of cloprostenol (60 ± 12.72% and 63 ± 2.22%). The conception rate in buffaloes were 45±6.91% and 55±6.91% in 500 µg and 875 µg of cloprostenol in single dose of PGF2a and 30±14.69% and 37±8.81% in 500 µg and 875 µg of cloprostenol in double dose of PGF2a. The calving rate in buffaloes were 10±5.20% and 15±0.79% in 500 µg and 875 µg of cloprostenol in single dose of PGF2a and 10±8.33 and 12±6.18% in 500 µg and 875 µg of cloprostenol in double dose of PGF2a. However, there were no significant differences in conception and calving rates in buffaloes neither between single and double doses PGF2a nor different concentrations of PGF2a. In conclusion, single dose of PGF2a with 875 µg of cloprostenol injection efficiently increased estrus synchronization rate in buffaloes.
 Bang. J. Anim. Sci. 2021. 50 (2): 80-85

Staphylococcus pasteuri (BCVME2) Resident in Buffalo Cervical Vaginal Mucus: A Potential Source of Estrus-Specific Sex Pheromone(s)

Mammals have microbes resident in their reproductive tract, some of which can be pathogenic while others may play a role in protecting the tract from infection. Volatile compounds play a role as sex pheromones that attract males for coitus during female estrus or heat. It is likely that these compounds themselves are secondary metabolites of bacterial flora resident in the vagina. In order to substantiate this hypothesis, bacteria were isolated from cervico-vaginal mucus (CVM) of buffalo during various phases of the estrous cycle and identified, using morphological, biochemical and molecular characteristics, asBacillusduring preestrus and diestrus, and as Staphylococcusduring all three phases of the estrous cycle. Populations of Staphylococcus differed between different phases of the estrous cycle, the predominant forms being S. warneri (BCVMPE1_1) during preestrus, S. pastueri(BCVME2) during estrus andS. epidermis (BCVMDE3) during diestrus. Mice were used as chemosensors to differentiate the estrus-specific S. pasteuri(BCVME2) from the others. Chemical analysis showed that S. pasteuri(BCVME2) produced acetic, propanoic, isobutyric, butyric, isovaleric and valeric acids. In addition, it was shown that S. pasteuri(BCVME2) volatiles influenced the sexual behaviors, flehmen and mounting, of the bull. Thus, S. pasteuri(BCVME2) is a potential source of vaginal pheromone(s) during estrus in buffalo.

Validation of salivary ferning based estrus identification method in a large population of water buffaloes (Bubalus bubalis) using Foldscope

Missing an estrus event is an economic problem in buffaloes because of lack of a simple and accurate estrus identification method. Saliva, a non-invasive fluid available every time, showed typical fern-like crystallization patterns at early estrus in buffaloes. However, to implement this salivary ferning based estrus identification method in the field conditions, the present study, for the first time, validated this method in four buffalo population samples (PS) representing four different field scenarios, an organized herd (PS1, 10 buffaloes monitored daily for a year (149 estrus events)), artificial insemination (AI) centers (PS2, 114 buffaloes brought for AI), induced estrus condition (PS3, 44 buffaloes) and farmers’ doorsteps (PS4, 275 random buffaloes with unknown reproductive history and no estrous signs). Totally, 582 saliva samples were collected from 443 buffaloes. Salivary smears were observed under a simple microscope and/or a less expensive (< 1USD) paper microscope, Foldscope. On the basis of salivary fern-like patterns, the proportions of estrus identification were significantly different among PS. Specifically, the proportions in the PS1 (0.74, P < 0.0001) and PS4 (0.08, P < 0.05) were significantly higher than their population proportion estimates, 0.5 and zero, respectively. Therefore, this estrus identification method is much useful in the PS1 and PS4, the practical field scenarios requiring an accurate estrus prediction method, compared to the PS2 and PS3. Especially, this method is 91 % confirmatory to detect early estrus in PS4. Therefore, salivary ferning is a useful technique to identify early estrus in buffaloes in the field conditions at farmers’ doorsteps.

Vascular Perfusion of the Preovulatory Follicle and its Relationship in Pregnancy Establishment during Natural and Induced Estrus in Buffaloes

Background: In buffaloes, little attention has been devoted on the haemodynamics of preovulatory follicle (POF) and serum estradiol profiles and their relationship with conception rate following AI at natural and induced estrus. Methods: Sixty healthy pleuriparous graded Murrah buffaloes were selected and divided into two groups, viz., Group I and II. Group I buffaloes were artificially inseminated twice at an interval of 24 hours during natural estrus. Estrus induction was done using PIVD and PGF2α in group II buffaloes and AI was performed 48 h and 72 h after the withdrawal of PIVD. The blood flow of the preovulatory follicle (POF) and early pregnancy were assessed in both the groups by Doppler ultrasonography.Result: On statistical analysis, the mean (± SE) of spectral indices revealed there was an increased blood flow to the POF during induced estrus (Group II) when compared to natural estrus (Group I). The conception rate observed was 66.67 and 58.33 per cent in group I and II, respectively. It is concluded that increased vascular perfusion to POF resulted in almost similar conception rates in induced and natural estrus following AI in buffaloes.

Molecular and functional characterization of buffalo nasal epithelial odorant binding proteins and their structural insights by in silico and biochemical approaches

The olfactory system is capable of detecting and distinguishing thousands of environmental odorants that play a key role in reproduction, social behaviours including pheromones influenced classical events. Membrane secretary odorant binding proteins (OBPs) are soluble lipocalins, localized in the nasal membrane of mammals. They bind and carry odorants within the nasal epithelium to putative olfactory transmembrane receptors (ORs). OBP has not yet been exploited to develop a suitable technique to detect oestrus which is being reported as a difficult task in buffalo. In the present study, using molecular biology and protein engineering approaches, we have cloned six novel OBP isoforms from buffalo nasal epithelium odorant-binding proteins (bnOBPs). Furthermore, 3 D models were developed and molecular-docking, dynamics experiments were performed by in silico approaches. In particular, we found four residues (Phe104, Phe134, Phe69 and Asn118) in OBP1a, which contributed to favourable interactions towards two sex pheromones, specifically oleic acid and p-cresol. We expressed this protein in Escherichia coli from female buffalo urine and validated through fluorescence quenching studies to show similar strong binding affinities of OBP1a to oleic acid and p-cresol. By using structural data, the binding specificity was also verified by site-directed mutagenesis of the four residues followed by in vitro binding assays. Our results enable us to better understand the functions of different nasal epithelium OBP isoforms in buffaloes. They also lead to improved understanding of the interaction between olfactory proteins and odorants to develop highly selective biosensing devices for non-invasive detection of oestrus in buffaloes. Communicated by Ramaswamy H. Sarma

Evaluation of pheromone-based kit: A noninvasive approach of estrus detection in buffalo.

In view of the silent nature of estrus in buffalo, a noninvasive assay kit has long been felt necessary for easy and effective estrus detection. This study was designed to detect estrus in buffalo using a kit formulated in our laboratory based on pheromone compound. Group I: Urine samples collected at estrus phase and group II: randomly collected urine samples were subjected to the test using the kit. No colour developed (i.e., positive reaction) in estrus urine after adding the kit solution. By contrast, pale and/or dark pink colour developed (i.e., negative reaction) in urine from the proestrus and diestrus buffaloes, respectively. Field evaluation of the kit in groups I and II revealed that 60.87% and 71.43% of urine samples were correctly identified as estrus and nonestrus (i.e., proestrus and diestrus), respectively. Therefore, the first of its kind estrus detection kit formulated based on urinary pheromone can as well be used as a simple device to detect estrus in buffalo.

Buffalo nasal odorant-binding protein (bunOBP) and its structural evaluation with putative pheromones

Pheromones are odoriferous volatile chemical cues produced by animals for communication among conspecifics so as to regulate their social behaviors. In general, the odor compounds are recognized by receptors in the nasal cavity. Odorant-binding protein (OBP), a lipocalin family protein, mediates the air-borne odor cues to nasal receptors through nasal mucus. The presence of OBP in several mammalian species is well documented but to-date there is no report of a nasal OBP in buffalo. Hence, the present study was undertaken to investigate if OBP is present in buffalo nasal mucus. Uni- and two-dimensional gel electrophoresis of the nasal mucus suggested the presence of OBP, which was confirmed using mass spectrometry. In silico homology model of the OBP was generated and its structural similarity with other mammalian OBPs was assessed. Finally, molecular-docking and -dynamics simulations analysis revealed the efficiency of buffalo nasal OBP (bunOBP) to bind with buffalo pheromones as well as other reported chemical cues. Taken together, the occurrence of nasal OBP in buffalo and its putative role in odor binding are reported for the first time. The potential association of this protein with estrus-specific volatiles could be taken to advantage for non-invasive detection of estrus in buffaloes.

Global proteomic analysis of water buffalo (Bubalus bubalis) saliva at different stages of estrous cycle using high throughput mass spectrometry

Accurate and efficient detection of estrus is one of the major constraints for exploitation of the production potential of buffalo owing to its poor manifestation of estrus signs, seasonal differences in expression and higher incidences of silent estrus (29%). The current study focused on identification of estrus specific candidate proteins in saliva of buffaloes. Estrus was detected based on behavioral signs in response to the teaser and changes in reproductive organs and confirmed by per-rectal examination, trans-rectal USG of reproductive organs, cervico-vaginal mucus characteristics and blood serum progesterone estimation. Day of onset of estrus was considered as day 0 and day −3, +3, +10 were considered as proestrus, metestrus and diestrus stage of the estrous cycle respectively. A total of 19 animals and their 38 estrous cycles (two from each) were included in this study. Saliva was collected from these animals during different stages of estrous cycle. Out of these, 08 animals were selected for global proteome analysis of saliva using in-solution digestion and nano-LC-MS/MS. A total of 275, 371, 304 and 565 proteins were identified with ≥2 peptides during proestrus, estrus, metestrus and diestrus stages of estrous cycle. Among the identified proteins 31, 62, 32 and 104 proteins were found specific to proestrus, estrus, metestrus and diestrus stage of the estrous cycle. Few salivary proteins such as Cullin-associated NEDD8-dissociated protein 1, Heat shock 70 kDa protein 1A, 17-beta-hydroxysteroid dehydrogenase type 1, Inhibin beta A chain, testin were identified as estrus specific and are important for estrus physiology. Taken together, these estrus specific proteins could be considered as the candidate biomarker for detection and confirmation of estrus in buffalo after thorough validation.

Effect of timing of artificial insemination in relation to onset of standing estrus on pregnancy per AI in Nili-Ravi buffalo.

The aim of the present study was to determine the optimum time of artificial insemination after the beginning of standing estrus in buffalo. Nili-Ravi buffalo (n = 109) during breeding season were exposed to teaser bull at 12 hours interval to determine the standing heat (0 h). Buffalo were randomly allocated to different time groups and a single artificial insemination was performed either at 0 h (n = 30), 12 h (n = 27), 24 h (n = 28) or 36 h (n = 24). In a subset of buffalo (n = 38) ultrasonography was performed, twice daily from 0 h (onset of standing heat) to determine the time of ovulation. Pregnancy diagnosis was performed 35-40 days after AI. Results revealed that mean time of ovulation from onset of standing heat was 34.7 ± 0.96 h (range 30 to 42 h). Higher (P < 0.05) pregnancy per AI were achieved in buffalo when inseminated at 24 h (15/28; 53%) compared to 0 h (8/30; 26%) and 36 h (3/24; 13%). Pregnancy per AI, was in-between, in buffalo, inseminated at 12 h (10/27; 37%) and did not differ (P > 0.05) with those bred either at 24 h or 0 h. The odds ratio further confirmed that the occurrence of pregnancy per AI was two times higher in buffalo inseminated at 24 h as compared to those at 12 h. It is concluded that optimal pregnancy per AI can be achieved when buffalo are bred artificially 24 h after the onset of standing heat.

Effect of Various Ovulation Synchronization Protocols on Estrus Response, Conception Rate and Blood Biochemical Profile in Anoestrus Buffaloes

This study evaluated the fertility response and plasma profile of biochemical and mineral constituents in 46 postpartum anoestrus (>90 days) buffaloes treated under field conditions with TriU-B/PRID, Ovsynch and Heatsynch protocols (n=12 each), keeping untreated anoestrus control (n=10) and normal cyclic control (n=10) groups. The estrus induction response with 3 treatment protocols was 83.33, 83.33 and 91.66 % respectively within 2-3 days of PGF 2 α injection. The conception rates (CRs) obtained at induced estrus in buffaloes under TriU-B, Ovsynch and Heatsynch protocols were 25.00, 33.33 and 25.00 %, and those of overall three cycles’ 50.00, 58.33 and 50.00 %, respectively, which were at par with normal cyclic control group (CR at first cycle 30.00 & overall of 3 cycles 50.00 %). Among untreated anoestrus control group, only two buffaloes (20%) exhibited estrus and conceived over 90 days follow up. The overall pooled mean plasma total cholesterol concentrations (mg/dl) in anoestrus buffaloes under TriU-B and in normal cyclic control group were the same (166.70±5.28 and 165.18±6.37) and significantly (P<0.01) higher than in Ovsynch (131.54±3.71) and lower than in Heatsynch (186.14±5.69) group. The value for total protein (g/dl) was significantly (P<0.05) lower in TriU-B (7.27±0.08) followed by other two groups (7.62±0.07 & 7.44±0.07) and highest in normal control group (7.98±0.10). The calcium was significantly higher (P<0.05) in Ovsynch (10.41±0.07 mg/dl) than other 3 groups (8.82±0.11 to 9.09±0.09 mg/dl), while inorganic phosphorus (3.99±0.10 to 4.29±0.13 mg/dl) did not vary between groups. Normal cyclic control buffaloes had significantly higher protein and lower calcium levels than in most treatment groups. The conceived buffaloes had apparently higher values of total cholesterol and protein as compared to non-conceived ones in all groups. In general, Ovsynch protocol was the best followed by TriU-B and Heatsynch for induction of estrus and improvement of fertility in anoestrus buffaloes.

Circulating levels of estradiol 17-β and progesterone vis à vis nitric oxide and nitric oxide synthase at and around oestrus in cycling buffaloes

The present study was designed to measure the circulating levels of estradiol 17-β and progesterone vis à vis nitric oxide (NO) and nitric oxide synthase (NOS) levels at and around oestrus in cycling buffaloes. Blood samples were collected −2, −1, 0, +1 and +2 days of oestrus cycle from cycling buffaloes and analyzed for NO, NOS, estradiol 17-β and progesterone levels. In present study, the levels of NO did not show significant (P < 0.05) variation from −2 day to day of oestrus then decreased significantly and ranged between 11.4 and 26.4 μM/L. The NOS concentration showed similar pattern as exhibited by NO in buffaloes and ranged from 0.56 to 1.28 U/L. The estradiol 17-β in buffaloes gradually increased and peaked at day of oestrus and ranged between 1.9 and 29.32 pg/ml and the pattern was observed almost similar to those of NO and NOS. The levels of progesterone decreased from −2 day to day of oestrus and then increased and ranged between 0.01 and 0.42 ng/ml. The present study generates data of NO, NOS, estradiol 17-β and progesterone at and around oestrus in buffaloes and that is of practical importance for clinical and experimental interpretations.

Exploration of salivary proteins in buffalo: an approach to find marker proteins for estrus.

Saliva is considered as the best source of biological material for biomarker discovery studies since it is noninvasive in comparison to other body sources. Usually buffalo cannot precisely express estrus signals. Hence, there is a need for concise methods to detect the time of estrus to ensure the success of artificial insemination. Therefore, we have established a reference proteome map on the whole saliva of buffalo during their estrous cycle with special reference to estrus. Nearly 12 bands have been observed using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of whole saliva. Collectively, 179 proteins are identified with respect to different phases of the estrous cycle using mass spectrometry. On the whole, 37 proteins are exclusively expressed in the estrus phase, which include β-enolase, Toll-like receptor (TLR) 4, clusterin, lactoperoxidase, serotransferrin, TGM3, UBA6, and transducin. Among the proteins, β-enolase and TLR 4 were validated, and their specific expression was found during estrus as compared to other phases using immunoblot. The functional annotation reveals many as binding proteins in the estrus saliva when compared to the other phases. The present findings conclude that the proteomic approach adopted to identify the proteins from buffalo saliva around the estrous cycle may provide a new tool for screening the estrus phase. The results further conclude that the specific expression of β-enolase and TLR 4 can be taken as the indicator of estrus in buffalo.

Faecal chemical cues in water buffalo that facilitate estrus detection

Chemo-signals are among the reliable non-invasive methods for estrus detection in mammals. Water buffalo is a silent heat animal and, hence, there is search for chemo-signals which would be effective non-invasive indicators of estrus state. We analyzed the faecal chemical cues during the estrous cycle in buffalo and to find the estrus-specific faecal volatile compounds adopting bull behavior assay. The faecal samples were collected at three phases of the estrous cycle (i.e., proestrus, estrus and postestrus) and subjected to gas chromatography–mass spectrometry analyses. We found 27 volatile compounds in the faeces of buffaloes, of which 4-methyl phenol (4mp) and trans-verbenol (tv) were found only in estrus faeces. The faecal samples of estrus buffaloes and the estrus-specific compound(s) (4mp+tv) at three different concentrations were tested for behavioral responses (flehmen and mounting behavior) in the bull. The bulls exhibited repeated flehmen when exposed to a combination of the two compounds (i.e., 4mp+tv) as compared to the individual compounds or raw faecal sample collected from buffalo when in estrus (P<0.05). However, higher number of mounting behavior was recorded when bulls were exposed to 4mp followed by a combination of the two compounds (4mp+tv) and trans-verbenol (P<0.05), in that order. By contrast, less number of mounting behavior was exhibited by bulls when exposed to the control sample (i.e., Hexadecanoic acid) (P<0.05). As inferred from the bull behavior assay, the present study suggests that the two compounds, 4 methyl phenol and trans-verbenol would be reliable indicators of estrus in buffaloes.

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 3-9year, 1st-6th lactations, were examined by oestrous detector and ultrasonographically for monitoring the ovarian and uterine activity for 7days 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±7.29pg/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.

Plasma progesterone, oestradiol‐17β and total oestrogen profiles in relation to oestrous behaviour during induced ovulation in Murrah buffalo heifers

The objectives of this study were to establish the characteristics of oestrous behaviour in Ovsynch (induction of ovulation through administration of GnRH-PGF2-GnRH in a systemic manner on 0, seventh and ninth day respectively) and Ovsynch plus Norprolac (Quinagolide hydrochloride – an inhibitor of prolactin secretion) treated Murrah buffalo heifers and to determine the relationships between this behaviour and the plasma concentrations of oestradiol-17β (E2), total oestrogen, and progesterone. Oestrus was detected by visual observations of oestrus signs, per rectal examination of genitalia and bull parading thrice a day during treatment period. Among all the symptoms, it was observed that bull mounting of heifers in oestrus was highest. Examination of genital tracts per rectum revealed that the cervix was relaxed, uterus was turgid and ovaries had palpable follicle in animals with oestrus. The peak concentrations of E2 (10.81 ± 0.62 pg/ml) and total oestrogen (17.11 ± 1.21 pg/ml) occurred at 9.45 ± 0.85 and 9.64 ± 0.93 h after second GnRH administration, respectively, in Ovsynch treated animals. However, the peak levels of E2 (20.02 ± 2.87 pg/ml) and total oestrogen (32.71 ± 3.15 pg/ml) occurred at 10.18 ± 0.50 and 10.36 ± 0.75 h after second GnRH administration, respectively, in Ovsynch plus Norprolac treated animals. Plasma progesterone concentration was basal (0.20 ± 0.001 ng/ml) during the peri-oestrus period. The plasma progesterone concentration was the lowest on the day of oestrus and increased to register a peak on day 13 ± 2 of the cycle. Oestrous behaviour was positively correlated with the peak concentration of E2 (p < 0.001) and total oestrogen (p < 0.001) during the peri-oestrus period. Inhibition of prolactin by Norprolac administration significantly increased the concentration of E2 and total oestrogen during oestrus in buffaloes in comparison to those recorded in animals subjected to Ovsynch protocol alone. In conclusion, our results suggest that the peak concentrations of E2 and total oestrogen and mean level of E2 and total oestrogen during the peri-oestrus period are the important factors contributing the behavioural manifestation of oestrus in buffalo cows.

Follicular Population during the Oestrous Cycle in Nili-Ravi Buffaloes Undergoing Spontaneous and PGF2a Induced Luteolysis

The objective of this study was to compare the follicular population during spontaneous and PGF 2α induced oestrous cycles in Nili-Ravi buffaloes. In Exp.1, (n = 13 oestrous cycles) follicular population was monitored using ultrasonography on alternate days. Buffaloes were monitored for ovarian follicles from day 0 (first oestrus) until next oestrus. These animals were observed for oestrus twice daily using a teaser bull. Of 12 oestrous cycles, 9 (75%) had two waves of follicular activity and only 3 (25%) had three waves during the oestrous cycle. The mean number of small, medium and large follicles among various days of the oestrous cycle between two and three waves of follicular development were not significantly different (p>0.05). In Exp. 2, follicular population 3 days before oestrus was compared in buffaloes undergoing spontaneous (n = 12 oestrous cycles) and PGF 2α induced (n = 6) luteolysis. The mean number of small and large follicles increased (p<0.05) and the number of medium follicles decreased (p<0.05) during the 3 days before oestrus in buffaloes undergoing induced luteolysis as compared to those with spontaneous luteolysis. These results showed that the mean number of small, medium and large follicles among various days of the oestrous cycle were similar between the two and three waves of follicular development, and three days before oestrous the number of small, medium and large follicles altered due to induced luteolysis on day 9, compared to those with spontaneous luteolysis.

Postpartum endocrinology and prospects for fertility improvement in the lactating riverine buffalo ( Bubalus bubalis) and yak ( Poephagus grunniens L.)

In many Asian countries the riverine buffalo is the major milk producing animal besides contributing towards draught power and meat production. The animal however suffers from inherent reproductive problems such as poor estrus expression and long calving intervals which limits its lifetime production. The yak is a seasonal breeder and the mainstay of highlanders and tribal populations inhabiting the inhospitable terrains of the Himalayan region. The factors responsible for its seasonality include poor nutrition, harsh climate and high altitude. Very little information is available on postpartum endocrinology in riverine buffaloes and even less so in yaks in relation to milk yield and cyclicity commencement. Our recent investigations on endocrine changes associated with cyclicity commencement in buffaloes (Murrah breed) showed a positive correlation between plasma prolactin concentrations and delay in postpartum cyclicity commencement. A significant correlation of plasma GH concentration with milk yield was also obtained. Monitoring cyclicity through milk progesterone analysis in buffaloes postpartum indicated that the incidences of silent estrus was low in winter months and very high in summer months — the overall annual mean being 37%. Preovulatory LH surges post-estrus occurred at different times resulting in ovulations at 28 to 60 h after onset of spontaneous estrus in buffaloes. Progesterone profiles in some yaks indicated cyclic activity even during non-breeding season. The positive correlation between plasma prolactin and melatonin indicates valuable evidence for their role in reproduction in this animal.