Effect of eCG dose on ovarian haemodynamics, hormonal profiles and prolificacy rate when oestrus was induced during low-breeding season in Beetal goats.

The objectives of the experiment were to determine the effect of two doses of equine chorionic gonadotropin (eCG) in a standard synchronization protocol based on a short-term progesterone (P4 ) priming on ovarian structures and haemodynamics, concentrations of steroid hormones and prolificacy rate when oestrus was induced during low-breeding season (LBS) in Beetal dairy goats. We hypothesized that inclusion of eCG in a short-term P4 priming-based synchronization protocol would increase the blood perfusion to ovarian structures leading to enhance oestrous and ovulatory responses and prolificacy rate in goats. Forty-two multiparous acyclic goats were blocked by body condition and, within block, assigned randomly to receive saline as control (CON), low eCG (L-eCG; 300 IU) or high eCG (H-eCG; 600 IU) dose. Initially, a controlled internal drug release (CIDR) device was placed in the anterior vagina on d -8, followed by removal of CIDR on d -3, concurrent with the administration of PGF2α and eCG according to their respective treatments. Goats were monitored for oestrous response. B-mode and Doppler ultrasonography was performed with 12-h interval, starting from day -3 until natural breeding (day 0), and then on days 5, 10, 15 and 20 post-breeding to monitor follicular and luteal dynamics and blood flow, respectively. Blood was sampled at 0, 12, 24, 36 and 60 h after CIDR removal to quantify plasma concentrations of estradiol-17ß (E2 ), whereas plasma concentrations of P4 were assayed at days 5, 10, 15 and 20 after breeding. Pregnancy and prolificacy rates were determined at day 30 and 150 after breeding, respectively. Data were analysed with mixed-effects models, and orthogonal contrasts were used to evaluate the effect of treatment [Con vs. (½ L-eCG + ½ H-eCG)] and dose of eCG (L-eCG vs. H-eCG). Data are presented in sequence as CON, L-eCG, H-eCG (LSM ± SEM). The oestrous intensity score (152.9 vs. 182.7 vs. 186.5 ± 15.1; p = .02) was greater in eCG-treated goats as compared to CON. Administration of eCG reduced the intervals to standing oestrus (66.2 vs. 41.8 vs. 48.9 h ± 5.5; p = .05), breeding (70.2 vs. 44.4 vs. 45.4 h ± 4.5; p = .03) and ovulation (84.5 vs. 61.2 vs. 63.4 h ± 6.2; p = .05) compared with CON goats. The mean growth rate of pre-ovulatory follicle was greater (1.11 vs. 1.49 vs. 1.45 mm ± 0.08; p = .01) in eCG-treated goats resulting in an increased diameter of pre-ovulatory follicle (6.27 vs. 7.20 vs. 7.31 mm ± 0.07; p < .01) and corpora lutea (6.75 vs. 8.26 vs. 8.07 mm ± 0.42; p = .04) than CON. The mean follicular blood flow did not differ among treatments; however, the mean luteal blood flow was greater in L-eCG-treated goats (0.81 vs. 1.61 vs. 1.07 cm2 ± 0.12; p = .001). The mean concentrations of E2 (4.03 vs. 5.21 vs. 4.78 pg/ml ± 0.42; p = .04) and P4 (4.85 vs. 6.39 vs. 6.22 ng/ml ± 0.34; p = .04) were greater in eCG-treated goats. The twinning rate did not differ between treatments; nevertheless, prolificacy rate was greater (p = .04) in L-eCG-treated goats. Collectively, our data suggest that the administration of eCG improves the induction of oestrous and ovarian dynamics. Administration of L-eCG enhances prolificacy rate, therefore, a low dose of eCG might be practically beneficial to improve reproduction during LBS in acyclic Beetal dairy goats.

Study on Various Luteal Characteristics Using Doppler Ultrasonography for Early Pregnancy Diagnosis in Nili-Ravi Buffaloes.

The objective of current study was to assess the trend in various luteal characteristics viz luteal size (LS), plasma progesterone (P4) concentration, and luteal blood flow (LBF) using color Doppler imaging (CDI) and power Doppler imaging (PDI) modes in pregnant and nonpregnant Nili-Ravi buffaloes. Lactating, cyclic, and healthy Nili-Ravi buffaloes (n = 09) without any reproductive abnormality were selected in present study. Buffaloes were synchronized using Ov-Synch, and fixed-time artificially insemination was performed (day = 0). Pregnancy was diagnosed on 30-day post-AI using B-mode ultrasonography based on presence or absence of embryonic heartbeat. Ovaries of all animals were scanned from day 5 till 21 post-AI using both B-mode and Doppler ultrasonography to measure LS and LBF. After each ovarian ultrasound examination, blood samples were collected via jugular venipuncture to determine plasma P4 concentration. According to results, LBF using CDI and PDI was significantly higher (P ≤ 0.05) in pregnant buffaloes on days 13 and 15 post-AI, respectively. The mean LS and plasma P4 concentration did not differ (P ≥ 0.05) between pregnant and nonpregnant animals until day 15 post-AI. However, a significant difference (P ≤ 0.05) was noticed for both on day 17 and onwards. It is concluded that LBF is a more sensitive luteal character as compared to LS and P4 for earlier pregnancy diagnosis in Nili-Ravi buffaloes when ascertained through CDI.

Vitrification improves in-vitro embryonic survival in Bos taurus embryos without increasing pregnancy rate post embryo transfer when compared to slow-freezing: A systematic meta-analysis.

Objectives were to use meta-analytic approaches to compare slow-freezing (SF) and vitrification (VF) methods of cryopreservation on in-vitro (n = 12,211) and in-vivo (n = 3473) survival of Bos taurus embryos. The literature was systematically reviewed and data from 40 manuscripts including 78 experiments, and comprising 183 treatment means, were used for the analyses. The in-vitro parameters included rates of re-expansion, hatching, and survival of blastocysts either at 24 h or 72 h post-thawing/warming and total number (TN) of embryonic cells, whereas in-vivo parameters evaluated pregnancy rate between 35 and 60 d post embryo transfer (ET). Mixed models were fitted using MIXED and GLIMMIX procedures of SAS. Additionally, classical meta-analytical statistics were also fitted using METAN and METAREG procedures of STATA. The final models included the fixed effects of methods of cryopreservation and random effects of the experiment. Rates (LSM ± SEM) of re-expansion (0.36 ± 0.07 vs. 0.48 ± 0.08), hatching (0.25 ± 0.05 vs. 0.42 ± 0.07), and survival (0.57 ± 0.09 vs. 0.76 ± 0.07) at 72 h post-thawing/warming were lower (P < 0.05) in SF than VF, respectively. The TN of embryonic cells (96.89 ± 7.15 vs. 117.83 ± 7.15) remained lower (P < 0.05) in SF than VF, however, the relative risk (RR) of pregnancy rate post ET remained similar (RR = 1.0, CI = 0.8-1.2; P > 0.05) between both methods. Collectively, VF technique has a short-term protective effect against cryodamage of preimplantation embryos, however, it might be dysregulating genes involved in pregnancy success post ET in cows.

Isolation and Characterization of Equine Uterine Extracellular Vesicles: A Comparative Methodological Study.

Extracellular vesicles (EVs) have been identified in the uterine fluid in different species and have been pointed as key players in the embryo-maternal dialogue, maternal recognition of pregnancy and establishment of pregnancy. However, little is known about the uterine EVs in the mare. Therefore, the present study aimed at characterizing EVs from uterine lavage of cyclic mares by comparing five EVs isolation methods and the combination of them: (1) ultracentrifugation (UC); (2) concentration of lavage volume by Centricon ultrafiltration (CE); (3) the use of CE with different washing steps (phosphate-buffered saline with or without trehalose); (4) size-exclusion chromatography with iZON-qEV columns, and (5) a combination of the methods with best results based on EVs yield, purity, and protein cargo profiles. Transmission electron microscopy and Western blotting confirmed the isolation of EVs by all methods but with quantitative and qualitative differences. Mass spectrometry provided differences in protein profiles between methods, number of identified proteins, and protein classes. Our results indicate that the combination of CE/trehalose/iZON/UC is an optimal method to isolate equine uterine EVs with good yield and purity that can be applied in future studies to determine the role of equine uterine EVs in embryo-maternal interactions.

Meta-analysis to determine effects of treatment with FSH when there is progestin-priming on in-vitro embryo production using ovum pick-up in Bos taurus cows.

Treatments with follicle stimulating hormone (FSH) to enhance ovarian follicular development before ovum pick-up (OPU) are important for improving in-vitro embryo production (IVEP) procedures in cows, however, their procedural efficacy needs to be evaluated. The objective of the present study was to use meta-analytic methods to determine the effects of FSH treatments prior to OPU when there is progestin-priming on ovarian functions and IVEP in parous Bos taurus cows (n = 243). The literature was systematically reviewed and data from eight experiments, with 23 treatment means including 448 OPU sessions, were used for analyses. All eight experiments included a group of cows in which there was no FSH treatment (CON), and treatment with FSH before OPU to conduct IVEP. Mixed models were fitted using MIXED and GLIMMIX procedures of SAS. Additionally, classical meta-analytical statistics were also fitted using METAN and METAREG procedures of STATA. Models included fixed effects of treatment (i.e., CON and FSH) and random effect of the experiment, and data were weighted using the inverse of the SEM squared to account for precision of each experiment. Number of medium sized follicles, quality of oocytes, and transferable embryos were greater (P < 0.05) in FSH-treated as compared with cows of the CON group, without indications of publication bias or heterogeneity of data. Taken together, the results from the present study lead to the recommendation for treatments with FSH prior to OPU for IVEP when there was progestin-priming imposed to obtain a large number of transferable embryos for economic sustainability of commercial embryo transfer programs in parous non-lactating Bos taurus cows.

Evaluation of haemodynamic changes of uterine arteries using Doppler ultrasonography during different stages of pregnancy in Bos indicus cows.

The objectives of the study were to evaluate haemodynamic changes and their relationships among ipsilateral (IPS) and contralateral (CONT) uterine arteries (UA) during different stages of pregnancy in Bos indicus cows. Multiparous pregnant cows (n = 40) having a gestation length 30.47 ± 0.54 (mean ± SD) days were randomly enrolled and subjected to Doppler ultrasonography sequentially at 1st, 2nd, 4th, 6th and 8th months of gestation. Blood flow indices including diameter of UA (mm), blood flow volume (BFVo, ml/min), blood flow velocity (BFVe, cm/s), time-averaged maximum velocity (TAMV, cm/s), pulsatility index (PI) and resistance index (RI) were recorded. Data were analysed with mixed models using the PROC MIXED procedures, and Pearson correlation coefficients were calculated using the PROC CORR statement in SAS. The final statistical models included the fixed effects of side of UA, gestation month and the interaction between side of UA and gestation month. Results revealed that the mean diameter of the UA (12.13 ± 0.22 vs. 10.09 ± 0.22), BFVo (1236.33 ± 0.55 vs. 770.41 ± 0.55), BFVe (17.18 ± 0.42 vs. 15.58 ± 0.42) and TAMV (17.11 ± 0.44 vs. 15.77 ± 0.44) was higher (p < .05) in IPS as compared to CONT side of the UA in cows. However, PI and RI did not differ between IPS and CON arteries of uterus in cows. A very high and positive correlation (r = .89; p < .05) existed between the diameter of UA and BFVo starting from 1st to 8th months of gestation in IPS as well as CONT sides of UA. Moreover, TAMV was highly and positively correlated (r = .91; p < .05) with BFVe throughout the gestation. In conclusion, these haemodynamic changes in the UA could be used as a valuable validity tool to differentiate the compromised pregnancy in Bos indicus cows.

Luteal blood flow measured by Doppler ultrasonography during the first three weeks after artificial insemination in pregnant and non-pregnant Bos indicus dairy cows.

The objective of this study was to determine if there are differences in luteal size (LS), progesterone (P4), and luteal blood flow (LBF) between pregnant and non-pregnant Bos indicus dairy cows during the first three weeks after insemination, and whether these parameters are related to each other. Lactating cows (n = 13) of mixed parity with a body weight of 430 ± 18 kg (mean ± SD), showing regular estrous cycle were used in the study. All cows were artificially inseminated and were classified as pregnant (embryonic heartbeat on day 30; n = 8) or non-pregnant (inter-estrus interval 17 to 21 days, n = 5). In order to compare the LS and LBF after artificial insemination, B-mode and color Doppler ultrasonography of ovaries were performed on days 4, 5, 6, 7 (first week), 8, 10, 12, 14, (second week), and 16, 17, 18, 19, 20, 21 (third week) in pregnant and non-pregnant cows. Results revealed that the mean LBF was consistently higher (P < 0.05) during days 7 through 21 in pregnant cows than in non-pregnant cows. The mean LS was higher (P < 0.05) on days 6 and 7, and from day 17 onwards, and the mean concentration of P4 was higher (P < 0.05) on days 19, 20, and 21 in pregnant cows. In conclusion, LBF is a more sensitive parameter than LS and P4 for detection of differences in luteal function between pregnant and non-pregnant Bos indicus dairy cows during the first three weeks after AI.

Equine chorionic gonadotropin (eCG) enhances reproductive responses in CIDR-EB treated lactating anovular Nili-Ravi buffalo during the breeding season.

The objective of this study was to evaluate the effect of equine chorionic gonadotropin (eCG) on the estrous response, ovulatory follicle development, ovulation rate, corpus luteum (CL) diameter, concentrations of progesterone (P4) and pregnancy per AI (P/AI) in CIDR-EB treated lactating anovular Nili-Ravi buffalo during the breeding season. Buffalo (n = 87), 60 to 200 Days postpartum were treated with CIDR at a random stage of the estrous cycle (Day 0) and assigned to two groups; with eCG (+eCG; n = 44), or without eCG (-eCG; n = 43). In both groups, each animal received 2 and 1 mg estradiol benzoate (EB) on Day 0 and then on Day 8, respectively. On Day 6, eCG and PGF2α were administered. On Day 7, CIDR were removed and FTAI was performed at 48 and 60 h later and pregnancy was diagnosed 35 Days post AI. The results revealed that eCG treatment increased (P <  0.05) the CL diameter on 15, 18, and 21 after the ovulation that resulted from imposing the estrous synchronization protocol than when eCG was not administered. Similarly, treatment with eCG resulted in an increased (P <  0.05) mean concentration of P4 as compared with animals not treated with eCG on Days 18 and 21 after the ovulation that occurred subsequent to imposing the estrous synchronization protocol. The ovulation rate was greater in eCG-treated buffalo as compared with those not treated with eCG (93% compared with 74%, respectively; P < 0.05). The P/AI was greater in buffalo treated with eCG than those not treated with eCG (56% compared with 31%, respectively; P < 0.05). In conclusion, the addition of eCG along with EB and P4 based treatments enhanced the estrous response, follicular and luteal function, ovulation rate, concentrations of P4, and P/AI in lactating anovular Nili-Ravi buffalo.

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 resynchronization with GnRH or progesterone (P4) intravaginal device (CIDR) on Day 23 after timed artificial insemination on cumulative pregnancy and embryonic losses in CIDR-GnRH synchronized Nili-Ravi buffaloes.

The objective of the present study was to determine the effect of resynchronization on Day 23 with either GnRH or P4 (controlled internal drug release device containing progesterone; CIDR) on pregnancy rate, cumulative pregnancy, and embryonic and fetal losses in CIDR-GnRH synchronized Nili-Ravi buffaloes. Buffaloes (n = 181) of mixed parity, lactating, 181 ± 73 days postpartum, a body condition score (BCS) of 3.2 ± 0.5 (scale of 1-5), and 450-600 kg weight were subjected to synchronization and resynchronization. All buffaloes received CIDR on Day -9.5. In addition, GnRH was injected 36 h after CIDR removal, and timed artificial insemination (TAI) was performed 18 h later (Day 0). On Day 23, buffaloes were randomly assigned to receive one of the following treatments: 1) CON (n = 63), 2) P4 (n = 55), and 3) GnRH (n = 63) for resynchronization (2nd AI). Pregnancy rate, and embryonic and fetal losses were monitored by serial ultrasonography on Days 30, 45, 60, and 90 after synchronization (1st TAI), respectively. The pregnancy rate in GnRH-treated buffaloes remained significantly and consistently higher (P < 0.05) than in the CON group at Days 30, 45, 60, and 90 after 1st TAI. Based on the pregnancy diagnosis, on Day 30 post 1st TAI, buffaloes that remained non-pregnant in the CON, P4, and GnRH groups received: 1) Artificial insemination on detected estrus (AIDE; n = 37), 2) CIDR-GnRH protocol (CIDR; n = 27), and 3) Ovsynch protocol (OVS; n = 23), respectively. The pregnancy rate in resynchronized buffaloes did not differ (P > 0.05) between the OVS and CIDR groups; whereas the, cumulative pregnancy rate in GnRH + OVS buffaloes (81%) after 1st and 2ndAI when determined on Day 64 was higher (P < 0.05) than that in CON + AIDE (59%) buffaloes. The embryonic losses were significantly lower (P < 0.05) in GnRH-treated (18%) buffaloes, than in CON (42%) buffaloes on Day 45 post 1st TAI. Fetal losses were fewer and did not differ (P > 0.05) due to treatments on Day 60 or 90 post 1st AI. In conclusion, 1) the pregnancy rate and cumulative pregnancy rate in GnRH-treated buffaloes were higher than in CON buffaloes on Day 64 after synchronization and resynchronization, and 2) embryonic and fetal losses were lower in GnRH-treated buffaloes than in CON buffaloes when determined from Day 31-90 post 1st TAI.

Evaluation of changes in blood flow of the uterine artery by Doppler ultrasonography during the estrous cycle in lactating Bos indicus cows.

The objective of this study was to evaluate the changes in uterine blood flow (UBF) based on the resistance index (RI) and the pulsatility index (PI) by Doppler ultrasonography throughout the estrous cycle in lactating Sahiwal cows (n=9). Cows were randomly selected during their spontaneous estrus. UBF was examined on alternate days in all cows during the estrous cycle. The results revealed that the mean RI values were higher (P<0.05) on day -1 (estrus) than on day 0 (ovulation). The mean RI values followed a consistent pattern on days 2, 4, 6, and 8 (metestrus to early diestrus) and were lower (P<0.05) than on days 10, 14 16, and 18 (diestrus to early proestrus). The RI and corpus luteum (CL) size negatively correlated during its static phase (r=-0.99; P<0.05). The mean RI was greater (P<0.05) in high producers than in low producing cows. The mean PI value was higher (P<0.05) on day 10 than on day -1 and day 0. Similarly, the mean PI values were lower (P <0.05) on days 2 and 4 than on day 10. The PI value remained significantly lower (P<0.05) on day 2 than on day 8 of the estrous cycle. There was a positive correlation between RI and P4 (r=0.70; P<0.05) and PI and P4 (r=0.56; P<0.05) during the estrous cycle. It is concluded that the RI of uterine arteries, as a measure of blood flow, is considerably lower, while the PI is substantially elevated during diestrus compared to estrus and ovulation in Sahiwal cows.

Effect of ovsynch versus prostaglandin F2α protocol on estrus response, ovulation rate, timing of ovulation and pregnancy per artificial insemination in Sahiwal cows.

Sahiwal cow is Bos indicus which is an important dairy breed of tropical and sub-tropical region. Research on reproduction is rare in this breed. The objectives of the present study were to determine the effect of Ovsynch (OVS) versus prostaglandin F2α (PG) protocol on estrus response and its intensity, ovulation rate, timing of ovulation and pregnancy per artificial insemination (AI) in Sahiwal cows. Experimental cows (n = 80) were of mixed parity, lactating, suckled, ≥120 days postpartum with body condition score 3.08 ± 0.34 and 375-475 kg of body weight which were randomly assigned to receive either OVS (n = 46) or PG (n =34) protocol. Cows were inseminated twice at 12 and 24 h after second gonadotropin releasing hormone in the OVS group, and 72 and 84 h after administration of prostaglandin F2α in PG group, respectively. The results revealed that estrus response did not differ (P > 0.05) and was 87% in OVS and 78% in PG cows. Ovulation rate did not differ (P > 0.05) and was 50% in both, OVS and PG cows. The pregnancy per AI did not differ (P > 0.05) and was 43% in OVS compared to 31% in PG cows. It is concluded that estrus response, ovulation rate and pregnancy per AI of OVS protocol is the same as PG in Sahiwal cows.