DYNAMICS OF ESTRUS BEHAVIOUR IN BUFFALOES: LOOKING AT VARIOUS REPRODUCTIVE ASPECTS RELATED TO ESTRUS AND METHODS FOR DETECTION

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.

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.

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.

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.

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

Despite the rapid expansion of in vitro embryo production (IVP) technology for genetic improvement in the cattle industry in the last decades, pregnancy rates by the transfer of IVP embryos are still lower than those derived from in vivo-produced embryos. The objective of this study was to analyse factors affecting pregnancy rates after the transfer of IVP Japanese Black cattle embryos under farm conditions. Holstein heifers (n = 4,475) and cows (n = 8,541) were selected as recipients. Most cows (80%) were managed in tie-stall barns and most heifers (80%) were managed in pens. Embryo transfers were performed for 9 years, from 2004 to 2012. The embryos were produced from oocytes derived from a local abattoir and semen from 14 proven bulls by the Livestock Improvement Association of Japan (Hamano and Kuwayama 1993 Theriogelogy 39, 703–712). The fresh IVP embryos (quality; IETS code 1) that reached the blastocyst stage after 7 to 8 days (insemination = Day 0) were transported by an airplane (2 h) and subsequently by a car (1.5 h). Embryos were non-surgically transferred to each recipient on Day 7 to 9 of their natural oestrous cycle on farms. Pregnancy was diagnosed on Day 40 to 60 after oestrus. Pregnancy results were statistically analysed using the GLM procedures of SAS. The following variables were included in the model: recipient parity (0, 1, 2, or 3), day (7, 8, or 9) of the oestrous cycle at the time of embryo transfer, oestrus behaviour (increased activity observed by farmers), presence of mucus at oestrus, presence of blood after oestrus, and year (1, 2, 3, 4, 5, 6, 7, 8, or 9) and season (April–June as spring, July–September as summer, October–December as fall, or January–March as winter) of embryo transfer. The Bonferroni correction was used to counteract the problem of multiple comparisons. Heifers had significantly higher pregnancy rates than cows (51.0% v. 37.9%, respectively; P &lt; 0.01), and first parity cows had higher pregnancy rates than third parity cows (42.9% v. 35.7%, respectively; P &lt; 0.01). Pregnancy rates were significantly higher in recipients that received an embryo transfer on Day 8 of their oestrous cycle, than on Day 7 (46.6% v. 42.4%, respectively; P &lt; 0.01). Recipients without oestrus behaviour had higher pregnancy rates than those with oestrus behaviour (46.3% v. 43.4%, respectively; P &lt; 0.01). The presence of mucus and/or blood after oestrus and the season of transfer were not found to significantly affect pregnancy rates. The results of this study indicated that performing IVP embryo transfers on Day 8 of a recipient’s oestrous cycle will improve the pregnancy rate, season does not have an effect on pregnancy rate, and the detection of oestrus by monitoring increased activity is not always reliable and instead should be determined by multiple symptoms on farm conditions.

BackgroundDeclining fertility is a major concern for dairy farmers today. One explanation is shorter and weaker expression of oestrus in dairy cows making it difficult to determine optimal time for artificial insemination (AI). Chemical communication is of interest in the search for tools to detect oestrus or to synchronise or enhance oestrous periods. Pheromones, used in chemical communication within species, can influence reproduction in different ways. The aim here was to investigate whether oestrous cycle length, and duration and intensity of oestrous expression in dairy heifers could be manipulated through exposure to pheromones in oestrual substances from other females.MethodsBeginning on day 16 of two consecutive control oestrous cycles, ten heifers of the Swedish Red Breed (SRB) were exposed to water. During the two following cycles the heifers were exposed to urine and vaginal mucus, obtained from cows in oestrus. Cyclicity parameters were monitored through hormone measurements, oestrus detection and ultrasonographic examination.ResultsWe found no difference in cycle length or in duration of standing oestrus between control and treatment. We did, however, find a tendency of interaction between type of exposure (control or treatment) and cycle number within type of exposure for cycle length (p = 0.068), with the length differing less between the treatment cycles. We also found a tendency of effect of type of exposure on maximal concentration (p = 0.073) and sum of concentrations (p = 0.063) of LH during the LH surge, with values being higher for the control cycles. There were also significant differences in when the different signs of oestrus occurred and in the intensity of oestrous expression. The score for oedema and hyperaemia of external genitalia was significantly higher (p = 0.004) for the control cycles and there was also a significant interaction between type of exposure and time period for restlessness (p = 0.011), with maximum score occurring earlier for treatment cycles.ConclusionsNo evidence of altered oestrous cycle length or duration of oestrus after exposure of females to oestrous substances from other females was found. Expression of oestrus, and maybe also LH secretion, however, seemed influenced by the exposure, with the effect of treatment being suppressive rather than enhancing.

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.

Detection of oestrus is a key determinant of profitability of dairy herds, but oestrus is increasingly difficult to observe in the modern dairy cow, with shorter duration and less intense oestrus. Concurrent with the unfavourable correlation between milk yield and fertility, oestrous detection rates have decreased to less than 50%. A number of mutations have been identified in genes associated with fertility and production traits, but, to date, no single nucleotide polymorphism (SNP) has been associated with oestrous expression. Therefore, the objective of this study was to investigate SNPs, linked to fertility, for the association with oestrous expression. Blood was collected from 205 Holstein Friesian dairy cows and genotyped at 41 loci of 18 genes chosen for their roles in the oestrous cycle and milk production. SNPs were then examined for correlations with increase in activity at oestrus, recorded via activity monitors, using generalised linear models. Physical activity increased at oestrus between two and four fold. Larger increases were associated with mutant alleles in oestrogen receptor-α and gonadotrophin releasing hormone receptor genes (P<0.05) and in the STAT5A gene (P<0.05). Smaller increases were associated with mutant alleles of the activin receptor type IIB and prolactin receptor genes (P<0.10). In conclusion, alleles in these five genes provide the opportunity for selection of animals displaying greater oestrous activity which could aid reversal of the decrease in oestrous detection and thereby contribute to sustainability of the dairy industry worldwide.

Features of follicle-stimulating hormone–stimulated follicles in a sheep model: keys to elucidate embryo failure in assisted reproductive technique cycles

Can ovariectomy be justified on grounds of behaviour?

Integrating an automated activity monitor into an artificial insemination program and the associated risk factors affecting reproductive performance of dairy cows

Control of follicular development is essential for estrous synchronization. This experiment determined differences in follicular development during synchronization in Brahman females. Cows (n=219) and heifers (n=90) in Fall (n=170) and Spring (n=139) were synchronized with a CIDR insert and administration of GnRH or PG treatment on d0 (1/2GnRH: n=106, GnRH: n=103, PG: n=100), and CIDR removal and PG administration on d6 followed by estrus detection and AI for 96h. Blood samples were collected on d0, d6, and d8 and/or AI to determine circulating progesterone (P4) concentrations. Transrectal ovarian ultrasonography occurred daily. Cows were grouped by CL presence (d0CL, d6CL) and P4 concentrations (d0P4, d6P4, d8P4, Low&amp;lt; 1 ng/mL, High≥1 ng/mL) on d0 and d6. The GLIMMIX procedure of SAS was used to analyze follicular wave initiation, wave emergence day, estrus expression, and pregnancy success. There were significant interactions of treatment by d0CL (P=0.0237), and season by d0P4 (P=0.0118) on wave initiation. Cows receiving 1/2GnRH with a CL on d0 (97±2%) and Fall cows having High d0P4 (96±3%) had the greatest probability of wave initiation. Age significantly impacted wave initiation (P=0.0213; heifers: 93±4%, cows: 79±5%). Interactions of treatment by season (P=0.0405) and treatment by d0CL (P=0.0379) significantly impacted wave emergence timing. Season (P&amp;lt; 0.0001; Fall: 23±5%, Spring: 65±6%), and the interactions of treatment by d0P4 (P=0.0291) and new wave by d0P4 (P=0.0357) influenced estrus expression. Cows receiving 1/2GnRH with High d0P4 and cows with High d0P4 that initiated a new wave had the greatest probability of estrus expression. Pregnancy rate differed by estrus expression (P=0.0055; no estrus=25±6%, estrus=54±8%) and semen type (P=0.0105; conventional=52±9%, sexed=26±6%), and tended to differ by d8P4 (P=0.0570; Low=30±7%, High=48±8%). These results suggest that differences in follicular development and behavioral estrus in synchronized Brahman females may be due to seasonality and endogenous progesterone production.

Impact of luteal blood perfusion and expression of estrus on pregnancy rates of Bos taurus embryo recipients exposed to fixed-time embryo transfer.

Sexual activities and oestrus detection in lactating Holstein cows