Supplementation with long-acting injectable progesterone 3 days after TAI impaired luteal function in buffaloes.

Three experiments were conducted to evaluate the effects of long-acting injectable progesterone (iP4) in buffalo cows. In Experiment 1, ovariectomized buffaloes received 300 mg (iP300) or 600 mg (iP600) of iP4, and serum P4 concentrations were evaluated. In experiment 2, three groups were compared: control or administration of 300 mg of iP4 3 (iP4-D3) or 6 days (iP4-D6) after timed artificial insemination (TAI). On day 16, reproductive tract was recovered for conceptus, endometrium, and corpus luteum (CL) analysis. In experiment 3, pregnancy per AI (P/TAI) and proportion of pregnancy losses were evaluated after administration of 300 mg of iP4 3 (iP4-D3) or 6 days (iP4-D6) after TAI in lactating buffaloes. In experiment 1, serum P4 concentrations remained over 1 ng/mL for ~ 3 days in both groups. The 300 mg dose was used in subsequent experiments. In experiment 2, CL weight and endometrial glands density were decreased, and conceptus length was increased in iP4-D3 compared to control and to iP4-D6 (P < 0.05). Transcript abundance of Prostaglandin F Receptor (FP) and ISG15 in CL and of ISG15 and MX1 in endometrium was greater in iP4-D3 when compared to control and to iP4-D6 (P < 0.05). In experiment 3, there was no difference among experimental groups for P/TAI at D30 and pregnancy losses (P > 0.1); however, iP4-D3 presented a lower P/TAI at day 60 (41.7%) when compared to control (56.8%) and iP4-D6 (57.7%; P = 0.07). In conclusion, administration iP4 at 3 days after TAI affects CL development and consequently decreases final pregnancy outcome in buffaloes.

Applying assisted reproductive technology and reproductive management to reduce CO2-equivalent emission in dairy and beef cattle: a review.

Methane emission from beef and dairy cattle combined contributes around 4.5-5.0% of total anthropogenic global methane. In addition to enteric methane (CH4) produced by the rumen, cattle production also contributes carbon dioxide (CO2) (feed), nitrous oxide (N2O) (feed production, manure) and other CH4 (manure) to the total greenhouse gas (GHG) budget of beef and dairy production systems. The relative contribution in standard dairy systems is typically enteric CH4 58%, feed 29% and manure 10%. Herds with low production efficiency can have an enteric CH4 contribution up to 90%. Digestibility of feed can impact CH4 emission intensity. Low fertility herds also have a greater enteric CH4 contribution. Animals with good feed conversion efficiency have a lower emission intensity of CH4/kg of meat or milk. Feed efficient heifers tend to be lean and have delayed puberty. Fertility is a major driver of profit in both beef and dairy cattle, and it is highly important to apply multi-trait selection when shifting herds towards improved efficiency and reduced CH4. Single nucleotide polymorphisms (SNPs) have been identified for feed efficiency in cattle and are used in genomic selection. SNPs can be utilized in artificial insemination and embryo transfer to increase the proportion of cattle that have the attributes of efficiency, fertility and reduced enteric CH4. Prepubertal heifers genomically selected for favourable traits can have oocytes recovered to produce IVF embryos. Reproductive technology is predicted to be increasingly adopted to reduce generation interval and accelerate the rate of genetic gain for efficiency, fertility and low CH4 in cattle. The relatively high contribution of cattle to anthropogenic global methane has focussed attention on strategies to reduce enteric CH4 without compromising efficiency and fertility. Assisted reproductive technology has an important role in achieving the goal of multiplying and distributing cattle that have good efficiency, fertility and low CH4.

Use of new recombinant proteins for ovarian stimulation in ruminants.

Currently, gonadotropin products (follicle stimulating hormone, FSH, and luteinizing hormone, LH) used in animal reproduction are produced by extraction and purification from abattoir-derived pituitary glands. This method, relying on animal-derived materials, carries the potential risk of hormone contamination and pathogen transmission. Additionally, chorionic gonadotropins are extracted from the blood of pregnant mares (equine chorionic gonadotropin; eCG) or the urine of pregnant women (human chorionic gonadotropin; hCG). However, recent advancements have introduced recombinant gonadotropins for assisted animal reproduction therapies. The traditional use of FSH for superovulation has limitations, including labor requirements and variability in superovulation response, affecting the success of in vivo (SOV) and in vitro (OPU/IVEP) embryo production. FSH treatment for superstimulation before OPU can promote the growth of a homogenous follicular population and the recovery of competent oocytes suitable for IVEP procedures. At present, a single injection of a preparation of long-acting bovine recombinant FSH (rFSH) produced similar superovulation responses resulting in the production of good-quality in vivo and in vitro embryos. Furthermore, the treatment with eCG at FTAI protocol has demonstrated its efficacy in promoting follicular growth, ovulation, and P/AI, mainly in heifers and anestrous cows. Currently, treatment with recombinant glycoproteins with eCG-like activity (r-eCG) have shown promising results in increasing follicular growth, ovulation, and P/AI in cows submitted to P4/E2 -based protocols. Bovine somatotropin (bST) is a naturally occurring hormone found in cows. Recombinant bovine somatotropin (rbST), produced through genetic engineering techniques, has shown potential in enhancing reproductive outcomes in ruminants. Treatment with rbST has been found to improve P/IA, increase donor embryo production, and enhance P/ET in recipients. The use of recombinant hormones allows to produce non-animal-derived products, offering several advantages in assisted reproductive technologies for ruminants. This advancement opens up new possibilities for improving reproductive efficiency and success rates in the field of animal reproduction.

Evaluation of clinical and subclinical endometritis impacts on the reproductive performance and milk production of dairy cows in Brazilian herds.

Clinical endometritis (CE) and subclinical endometritis (SE) are two of the main uterine diseases in dairy cows during the puerperium period that can directly affect milk production and fertility in dairy herds. The first objective of this study was to investigate the effects of the CE and SE on the subsequent reproductive performance and milk production of dairy cows in Brazilian herds, and the second objective was to evaluate the reproductive performance and milk production of dairy cows according to the positive or negative isolation of bacteria in the uterine environment. A total of 279 lactating Holstein dairy cows (28-33 days in milk) from six commercial farms were studied. These were classified into three groups: healthy cows (without CE and SE, n = 161), cows with CE (vaginal discharge score = 3 and ≥18% PMNL, n = 83) and cows with SE (absence of signs of CE and >18% PMNL, n = 35). According to the presence of bacteria isolated from the uterine environment, the animals were classified as healthy cows (n = 161), CE cows with positive (n = 68) and negative (n = 15) bacterial isolation; and SE cows with positive (n = 21) and negative (n = 14) bacterial isolation. Cows with CE (20.5%) and SE (31.4%) had a lower (p < .05) conception rate at first AI than healthy cows (47.8%). Furthermore, both cows with CE (3.1) and SE (2.8) needed more service to become pregnant than healthy cows (1.7) and more days to be pregnant (50.3 days in CE and 43.1 days in SE than healthy group; p < .001). Cows with CE (23.8 kg) and SE (23.7 kg) produced less milk than healthy cows on the day of sampling (28.2 kg); however, milk production during 305 DIM of cows with CE and SE was not impaired when compared to healthy cows. The logistic regression showed that parity, body condition score, 305 days milk production, last calving (eutocia or dystocia), twin birth and stillbirth were not associated with the incidence of CE and SE. In addition, we highlight that cows with CE and SE, but with negative bacteria isolation, did not have reproductive performance and milk production impaired during the postpartum period. These results support that the use of antibiotics would not be beneficial in these cows, contributing to the strategic and rational use of antibiotic therapy on dairy farms.

The use of herbal treatments as alternatives to control uterine diseases in dairy cows.

Uterine diseases are a common gynecological issue that affects dairy cows during the postpartum period. The treatment of uterine diseases is mainly managed through antimicrobials; however, the problem of antibiotic residues, increased antimicrobial resistance of pathogens, and the abuse of antibiotics causes serious problems in the dairy chain around the world, which can affect the efficiency of conventional medicines. In these regards, alternative treatment strategies are needed to reduce the use of antibiotics in dairy production. This review primarily provides an overview that focuses on various alternative sources and methodologies, including plants extracts and essential oils, for the control of bovine uterine diseases, and it presents the advantages and limitations related to the use of these herbal therapies.

Productive and reproductive performance of crossbred dairy heifers with induced lactation and efficacy of antimicrobial therapy associated with internal teat sealants / Desempenho produtivo e reprodutivo de novilhas com lactação induzida e eficácia da terapia antimicrobiana associada com selante interno de tetos

This study evaluated (a) the efficacy of an association between injectable antibiotic therapy and sealant (ATBS) on milk yield (MY), somatic cell count (SCC), and prevalence of intramammary infections (IMI); and (b) the efficacy of gonadotropin-releasing hormone (GnRH) on follicular cyst (FCs) resolution (cyclicity at the 45th day in milk; DIM) and cumulative pregnancy rate (CPR) in heifers submitted to a lactation induction protocol (LIP). A total of 114 crossbred (Holstein × Jersey) heifers, with 34.7 ± 4.8 months and 439 ± 56.35 kg were submitted to LIP. On the 5th day of the LIP, the heifers were assigned to (i) ATBS (n = 57) with 7 mg/kg of norfloxacin associated with sealant and (ii) Control 1 (n = 57; CONT1) with no treatments. Lactation began on the 21st day of LIP and the 15th DIM, FCs were diagnosed and 106 heifers were randomized into two treatment groups with 53 heifers each: (i) GnRH (5 mL injectable GnRH) and (ii) Control 2 (CONT2; no treatment). Of the 114 heifers initially induced, 83.33% (n = 95) responded to LIP with an average MY of 15.19 kg/milk/day during 22 weeks of lactation. In the first 14 DIM, the IMI prevalence was 18% and 28% for heifers ATBS and CONT1 treated, respectively. Additionally, coagulase-negative Staphylococcus was the most frequently isolated group of pathogens. Mammary quarters that received ATBS treatment had a lower risk of IMI and SCC than CONT1. The cyclicity at 45 DIM was 68% (ATBS) and 35% (CONT1), and 57% and 46% for animals in the GnRH and CONT2. CPR was 60% in the ATBS group and 89% in CONT1, but GnRH treatment did not affect the CPR. In conclusion, LIP was effective in stimulating MY in heifers, and the IMI prevalence decreased with ATBS treatment. Also, the use of GnRH did not affect the FC regression, cyclicity at 45 DIM, and CPR.(AU)

Este estudo avaliou a (i) eficácia da associação entre antibioticoterapia injetável e selante interno de tetos (ATBS) na produção de leite (PL), contagem de células somáticas (CCS), e prevalência de infecções intramamárias (IIM); e (ii) eficácia do hormônio liberador de gonadotrofina (GnRH) na resolução de cistos foliculares (CFs), ciclicidade ao 45º dia em lactação (DEL) e taxa de prenhez cumulativa (TPC) em novilhas submetidas a um protocolo de indução de lactação (PIL). Um total de 114 novilhas mestiças (Holandês × Jersey), com 34,7 ± 4,8 meses e 439 ± 56,35 kg foram submetidas ao PIL. No 5º dia do PIL, as novilhas receberam: (i) ATBS (n = 57) com 7 mg/kg de norfloxacina associada ao selante interno de tetos e (ii) Controle 1 (n = 57; CONT1) sem tratamento. A lactação teve início no 21º dia do PIL e no 15º DEL, foram diagnosticados CFs e 106 novilhas foram agrupadas em dois grupos de tratamento com 53 novilhas em cada: (i) GnRH (5 mL de GnRH injetável) e (ii) Controle 2 (CONT2; sem tratamento). Das 114 novilhas inicialmente induzidas, 83,33% (n = 95) responderam ao PIL com PL média de 15,19 kg/leite/d durante 22 semanas de lactação. Nos primeiros 14 DEL a prevalência de IIM foi de 18% e 28% para as novilhas tratadas com ATBS e CONT1, respectivamente. Além disso, estafilococos coagulase negativa foram o grupo de patógenos mais frequentemente isolados. Quartos mamários tratados com ATBS tiveram menor risco (0,56) de IIM e menor CCS do que CONT1. A ciclicidade a 45 DEL foi de 68% (ATBS) e 35% (CONT1), e 57% e 46% para os animais no GnRH e CONT2. A TPC foi de 60% no grupo ATBS e 89% no CONT1, porém o tratamento com GnRH não afetou a TPC. Em conclusão, o PIL foi eficaz em estimular a PL em novilhas tardias e a prevalência de IIM diminiuiu com o tratamento ATBS. Além disso, o uso de GnRH não afetou a regressão de CF, ciclicidade em 45 DEL e a TPC.(AU)

Sperm function and oxidative status: Effect on fertility in Bos taurus and Bos indicus bulls when semen is used for fixed-time artificial insemination.

Semen quality is one of the criteria used for the selection of bulls with relatively greater fertility. In addition, bull fertility depends on the integrity and function of all sperm structures. The aim of this study, therefore, was to determine associations when there was conducting of conventional and functional techniques for the evaluation of sperm samples from bulls with known fertility history as determined when semen from these bulls was used for fixed-time artificial insemination programs. The study was designed in a 2 × 2 factorial arrangement, with one factor being breed (Angus x Nellore) and the other fertility (greater x lesser). Greater fertility groups were composed of ten Angus and 11 Nellore bulls, while lesser fertility groups were composed of ten Angus and seven Nellore bulls. Sperm were analyzed, in four cryopreserved distinct batches for each animal, for morphology, kinetics, plasma and acrosomal membrane integrity, mitochondrial activity and mitochondrial membrane potential, DNA integrity and oxidative status. There was no difference in characteristics commonly used in sperm quality conventional analysis. The results from functional analysis indicated an important association between mitochondrial dysfunctions, oxidative stress, and damage to sperm structures in lesser fertility bulls. Greater fertility bulls had greater sperm quality and indicators of functional cell structures. The associations, when there were evaluations using different techniques, indicate the importance of evaluation and correlation between different sperm functions to understand effects of distinct parameters on sperm fertilization capacity.

Lack of effect of melatonin on ovarian function and response to estrous synchronization and fixed-time AI during the nonbreeding season in lactating dairy buffalo (Bubalus bubalis).

The present study was conducted to examine whether pretreatment with melatonin would enhance ovarian follicular functions and increase response to estrous synchronization and fixed-time AI (TAI) during the nonbreeding season in lactating dairy buffalo. In Experiment 1, buffalo cows without a detectable corpus luteum (CL) were assigned on Day -20 (D-20) to three groups: control (n = 12); melatonin (n = 13); progesterone (P4) (n = 15). Cows in the melatonin group were implanted with melatonin on D-20. From D0 to D9, there was imposing of an estrous synchronization treatment regimen using either a standard Ovsynch protocol (control, melatonin) or a P4-based Ovsynch treatment regimen (P4). There were no differences (P > 0.05) among groups for the presence of a CL at D0, size of the largest follicle at D0, ovulation to GnRH injection at D0 and D9, or the time to ovulation after injection of GnRH at D9. In Experiment 2, there was imposing of the same treatment regimens as in Experiment 1, with inclusion of TAI. Females of the P4 group had a greater (P = 0.001) pregnancy/AI percentage (60 %) than those in the control (17 %) and melatonin (23 %) groups. Females of the P4 group also had a larger (P = 0.005) CL at D20 compared with those in the control and melatonin groups. Findings indicate treatment with melatonin for 20 days did not affect ovarian functions or the response to an estrous synchronization treatment regimen and TAI during the nonbreeding season in lactating dairy buffalo.

Estradiol Priming Potentiates the Kisspeptin-Induced Release of LH in Ovariectomized Cows.

The present study examined whether priming with estradiol benzoate (EB) for 12 h increased both the peak and duration of LH release in response to kisspeptin (KISS1, KP) in cows. In a Latin square design, ovariectomized Nelore cows (n = 8) received: Control, i.m. 4 mL of 0.9% saline; KP, i.m. 4 mg murine KISS1-10; EBKP, i.m. 4 mg KISS1-10 + i.m. 2 mg EB simultaneously; EB12KP, i.m. 4 mg KISS1-10 + i.m. 2 mg EB 12 h before KISS1-10. Concentrations of LH were determined in blood samples obtained at time 0 (treatment), 20, 40, 60, 90, 120, 150, 180, 210 and 270 min. Concentrations of LH were analyzed by Proc GLIMMIX for repeated measures. In case of significance, the adjusted Tukey test was used to test for differences among treatments. GraphPad 8.0 PRISM® was used to determine the area under the LH-response curve (AUC) after injection of KISS1-10. Plasma LH remained relatively constant throughout sampling after treatment with saline. The peak in LH after injection of KISS1-10 occurred at 20 min in Groups KP and EBKP and at 40 min in Group EB12KP. The peak LH response (∆LH, ng/mL) was greater (p < 0.01) in Group EB12KP (5.6 ± 0.9) than in Groups KP (2.4 ± 0.9) and EBKP (3.5 ± 0.9), which did not differ. AUC (LH ng/mL*min) was greater (p = 0.02) in Group EB12KP (439 ± 73) than in Groups KP (176 ± 73) and EBKP (241 ± 73), with the latter two groups not differing. The findings indicated that 12 h priming with EB increased both the peak and duration of the LH response to treatment with KISS1. The incorporation of EB priming and KISS1 could improve the efficiency of estrus synchronization with fixed-time AI in cows. This would have an important practical application in assisted breeding in beef and dairy cattle.

Feasibility and accuracy of using different methods to detect pregnancy by conceptus-stimulated genes in dairy cattle.

Development of new methods for early diagnosis of pregnancy can be important to increase the reproductive efficiency and profitability of dairy herds. The bovine conceptus secretes IFN-τ that stimulates the transcription of several genes in circulating immune cells and extrauterine tissues. The aims of this study were to evaluate the mRNA abundance for pregnancy predictability of a classic gene stimulated by IFN-τ (ISG15) and a novel potential pregnancy marker (LGALS3BP) in peripheral blood mononuclear cells (PBMC), total blood leukocytes (TBL) or milk leukocytes (TML), and cervical cells (CC) on d 20 after timed artificial insemination (TAI) in dairy cattle. Eighteen Holstein females (12 cows and 6 heifers) were submitted to an estrous synchronization protocol for TAI (d 0). On d 20 post-TAI, blood samples were collected from coccygeal vessels for isolation of PBMC and in Tempus Blood RNA tubes (Applied Biosystems) for TBL. Samples of CC were collected using a cytological brush, and the TML were isolated from milk samples collected before routine milking. Pregnancy diagnosis was performed on d 30 post-TAI using transrectal ultrasonography, and females were classified as pregnant (n = 8) or nonpregnant (n = 10). Total RNA was extracted and mRNA abundance of target genes (ISG15 and LGALS3BP) was quantified by reverse-transcription quantitative PCR and normalized in relation to reference genes. Data were analyzed by ANOVA using the MIXED procedure of SAS (SAS Institute Inc.). The mRNA abundance of ISG15 was greater in pregnant than in nonpregnant animals for PBMC, TBL, and CC. No difference was detected for TML based on pregnancy status. For LGALS3BP mRNA abundance, no difference was detected between pregnant and nonpregnant animals for PBMC, TBL, and TML, but a tendency for greater abundance in pregnant animals was observed for CC. The fold change for ISG15 in each pregnant cow related to the mean of nonpregnant animals was 2.73 ± 0.31, 3.40 ± 2.17, 1.64 ± 0.29, and 0.005 ± 0.002 for PBMC, CC, TBL, and TML, respectively. The fold change for LGALS3BP in each pregnant cow related to the mean of nonpregnant animals was 0.97 ± 0.38, 1.77 ± 0.39, 0.20 ± 0.08, and 0.70 ± 0.11 for PBMC, CC, TBL, and TML, respectively. The receiver operating characteristic curve analysis indicated that ISG15 abundance predicted pregnancy in PBMC (area under curve, AUC = 0.92) and CC (AUC = 0.77) but not in TBL (AUC = 0.72) or TML (AUC = 0.52). In conclusion, mRNA abundance for ISG15 in PBMC was the best predictor for pregnancy at d 20 post-TAI, whereas TBL and TML were not good predictors of pregnancy on d 20 post-TAI. The mRNA abundance of LGALS3BP was not associated with pregnancy status in any type of cell evaluated.

"Which Factors Affect Pregnancy Until Calving and Pregnancy Loss in Buffalo Recipients of in vitro Produced Embryos?"

In vitro embryo production and embryo transfer (ET) in buffaloes has been developed for decades. However, most studies are focused on the donor or laboratory improvements, and there is a lack of reports regarding the recipients. Therefore, our aim was to investigate factors associated to pregnancy (P/ET), pregnancy loss (PL), and calving rates in buffalo recipients. The studied factors were season, recipient parity, the synchronization protocol, the CL diameter, asynchrony between the embryo and the recipient, the day of the recipient estrous cycle, the embryo (fresh vs. vitrified), the day of embryo development, and the embryo stage. These retrospective data, from a program of in vitro produced embryos, were analyzed by logistic regression, and the odds ratio was also estimated. Two factors were related to P/ET and the calving rate: (1) progesterone associated to estradiol plus eCG protocol for fixed time ET tended to affect positively P/ET on day 30 (41.9 vs. 36.1%, respectively; P = 0.07; AOR = 1.28) and P/ET on day 60 (37.8 vs. 36.1%, respectively; P = 0.09; AOR = 1.08) compared to the Ovsynch protocol; and (2) the CL diameter (≥14.5 mm) at transfer increased P/ET on day 30 (47.4 vs. 32.5%; P < 0.01; AOR = 1.87) and on day 60 (45.3 vs. 27.7%; P < 0.01; AOR = 2.16), and also the calving rate (37.9 vs. 21.7%; P < 0.01; AOR = 2.20). PL was greater when ET was done in the nonbreeding season compared to the breeding season (PL 30-60: 12.8 vs. 0.0%, P = 0.01; AOR > 999.99; PL 60-calving: 26.8 vs. 3.6%, P = 0.03; AOR = 9.90; and PL 30-calving: 36.2 vs. 3.6%, P = 0.01; AOR = 15.30). In conclusion, the data of our study indicated that the synchronization protocol, the CL diameter, and ET during the breeding season impacted the reproductive efficiency of buffalo recipients.

Ovarian follicular dynamics, progesterone concentrations, pregnancy rates and transcriptional patterns in Bos indicus females with a high or low antral follicle count.

We evaluated the effect of the antral follicle count (AFC) on ovarian follicular dynamics, pregnancy rates, progesterone concentrations, and transcriptional patterns of genes in Nelore cattle (Bos taurus indicus) after a timed artificial insemination (TAI) programme. Cows were separated based on the AFC, and those with a high AFC showed a larger (P < 0.0001) ovarian diameter and area than those with a very low AFC. Females with a very low AFC exhibited a larger (P < 0.01) diameter of the dominant follicle at TAI (13.6 ± 0.3 vs. 12.2 ± 0.4 mm) and a tendency (P = 0.06) to have different serum progesterone concentrations (2.9 ± 0.3 vs. 2.1 ± 0.3 ng/mL; on day 18, considering day 0 as the beginning of the synchronization protocol) than those with a high AFC. The pregnancy rate was higher (P ≤ 0.05) in animals with a very low (57.9%) and low (53.1%) AFC than in those with a high AFC (45.2%). The expression of genes related to intercellular communication, meiotic control, epigenetic modulation, cell division, follicular growth, cell maintenance, steroidogenesis and cellular stress response was assessed on day 5. In females with a low AFC, 8 and 21 genes in oocytes and cumulus cells, respectively, were upregulated (P < 0.05), while 3 and 6 genes in oocytes and cumulus cells, respectively, were downregulated. The results described here will help elucidate the differences in ovarian physiology and the reproductive success of Bos indicus females with a low or high AFC.

Treatment with estradiol cypionate at progesterone withdrawal reduces handling without compromising the pregnancy rate to timed-AI in buffalo.

The aim of this study was to determine if treatment with estradiol cypionate (EC) at the time of P4 withdrawal induced ovulation in a synchronization/timed-AI (TAI) protocol in buffalo. In Experiment 1, 56 buffaloes received an intravaginal P4 device (1.0 g) plus estradiol benzoate (EB, 2.0 mg im) on Day 0 (D0). On Day 9, the P4 device was removed and buffaloes were given PGF2α (0.53 mg im sodium cloprostenol) plus eCG (400 IU im). Buffaloes were then randomly allocated to one of two groups: Group GEC (n = 29), treated with EC (1.0 mg im) at P4 device removal; Group GEB (n = 27), treated with EB (1.0 mg im) 24 h after P4 device removal. Ovarian ultrasound was undertaken on: D0, to ascertain general ovarian status; D9 to D11 (every 24 h), to measure diameter of the largest follicle (LF) and follicular growth rate; D11 to D13 (every 12 h for 72 h), to determine the time of ovulation and ovulation rate. Following P4 device removal, Groups GEC and GEB had a similar follicular growth rate (0.9 ± 0.1 and 1.1 ± 0.1 mm/day, respectively; P = 0.15) and similar LF diameter on D11 (11.4 ± 0.6 and 12.5 ± 0.5 mm; P = 0.12). Groups GEC and GEB also had a similar diameter of the ovulatory follicle (13.0 ± 0.5 and 13.4 ± 0.6 mm; P = 0.52), interval from P4 device removal to ovulation (68.2 ± 2.8 and 71.1 ± 1.4 h; P = 0.41) and ovulation rate (62.1% and 70.4%; P = 0.44). In Experiment 2, 199 buffaloes were assigned to the two treatments in Experiment 1 (GEC, n = 100; GEB, n = 99). All animals underwent TAI 56 h after P4 device removal and pregnancy diagnosis was preformed on D41. The pregnancy rate was similar for Groups GEC and GEB (50.0 and 45.5%, respectively; P = 0.45). The findings indicate that treatment with EC at the time of P4 withdrawal induces ovulation and achieves the same pregnancy rate to TAI as treatment with EB 24 h after P4 removal. The use of EC requires one less handling which is highly important in facilitating practical adoption of TAI in assisted breeding and genetic improvement in buffalo.

Genetic Parameters and Genome-Wide Association Studies for Anti-Müllerian Hormone Levels and Antral Follicle Populations Measured After Estrus Synchronization in Nellore Cattle.

Reproductive efficiency plays a major role in the long-term sustainability of livestock industries and can be improved through genetic and genomic selection. This study aimed to estimate genetic parameters (heritability and genetic correlation) and identify genomic regions and candidate genes associated with anti-Müllerian hormone levels (AMH) and antral follicle populations measured after estrous synchronization (AFP) in Nellore cattle. The datasets included phenotypic records for 1099 and 289 Nellore females for AFP and AMH, respectively, high-density single nucleotide polymorphism (SNP) genotypes for 944 animals, and 4129 individuals in the pedigree. The heritability estimates for AMH and AFP were 0.28 ± 0.07 and 0.30 ± 0.09, and the traits were highly and positively genetically correlated (rG = 0.81 ± 0.02). These findings indicated that these traits can be improved through selective breeding, and substantial indirect genetic gains are expected by selecting for only one of the two traits. A total of 31 genomic regions were shown to be associated with AMH or AFP, and two genomic regions located on BTA1 (64.9-65.0 Mb and 109.1-109.2 Mb) overlapped between the traits. Various candidate genes were identified to be potentially linked to important biological processes such as ovulation, tissue remodeling, and the immune system. Our findings support the use of AMH and AFP as indicator traits to genetically improve fertility rates in Nellore cattle and identify better oocyte donors.

Embryo transfer in buffalo (Bubalus bubalis).

The use of assisted reproductive technologies, such as superovulation and in vivo embryo production and in vitro embryo production (IVEP), has increased rapidly in recent years and is now applied worldwide for genetic improvement in beef and dairy buffaloes. Although in vivo embryo production has been shown to be feasible in buffalo, low efficiency and limited commercial application has been documented. These results could be associated with low antral follicle populations, high levels of follicular atresia and/or failures of the oocyte to enter the oviduct after superovulation. Additionally, IVEP technology has been shown to be an important tool for multiplying genetic material from donors of superior merit, and promising results have been achieved with the use of ovum pick-up (OPU) along with IVEP in buffalo. However, several factors appear to be critical for successful OPU/IVEP, including circulating levels of anti-Müllerian hormone, antral follicle populations, sizes of the follicles available for the OPU, reproductive seasonality, semen (sire) used for IVEP, donor category and farm. Furthermore, technologies applied to control follicular wave emergence and ovulation at predetermined times, without the need for estrus detection in recipients, has facilitated management and improved the efficiency of embryo transfer programs in buffalo herds. Conclusively, with the considerable evidence of poor results with in vivo embryo production in buffaloes, the association of OPU with IVEP represents a new alternative for the exploitation of buffalo genetics.

Administration of PGF2α during the periovulatory period increased fertilization rate in superovulated buffaloes.

The aim of the present study was to determine the recovery of embryonic structures (ova/embryos) and fertilization rate in superovulated buffaloes treated with PGF2α during the periovulatory period. On day 0 (D0), buffaloes at random stages of the estrous cycle were treated with an intravaginal progesterone device (P4; 1.0 g) and estradiol benzoate (EB, 2.0 mg i.m.). From D4 to D7, all buffaloes received i.m. FSH (200 mg total) twice-daily over 4 days in decreasing doses. On D6 and D7, the animals were given PGF2α analogue (0.53 mg i.m. sodium cloprostenol) and the P4 device was removed on D7. On D8, all buffaloes received GnRH (20 µg i.m. buserelin acetate). Buffaloes were then randomly allocated to one of three groups: control (Group C, n = 18), no further treatment; PGF2α analogue injection (Group IM-PGF; n = 18), four injections (0.53 mg i.m. sodium cloprostenol) 12 h apart, from D8 to D10; PGF2α analogue osmotic pump (Group OP-PGF; n = 18), s.c. osmotic mini-pump (2.12 mg sodium cloprostenol) from D8 to D10. The study had a crossover design (three treatments x three replicates). All animals underwent timed AI, 12 and 24 h after treatment with GnRH. Embryonic structures were recovered on D14. Ovarian ultrasonography was used on D8 and D14 to record follicular superstimulation and superovulatory responses. Blood samples were obtained on Days 7, 8, 9 and 10 to measure circulating concentrations of P4, E2 and PGFM. Data were analyzed by GLIMMIX procedure of SAS®. There was no effect (P = 0.58) of treatment on the total number of embryonic structures (Group C, 2.1 ±â€¯0.8; Group IM-PGF, 2.1 ±â€¯0.6; Group OP-PGF, 1.4 ±â€¯0.4). There was also no effect (P = 0.93) of treatment on the recovery rate of embryonic structures (oocytes and embryos D14/CL D14). The fertilization rate was higher (P = 0.04) in Groups IM-PGF (84.6%) and OP-PGF (88.0%), which did not differ, than Group C (63.2%). The viable embryos rate was greater (P < 0.01) for Groups IM-PGF (82.0%) and OP-PGF (88.0%) than Group C (52.6%). There was no interaction between treatment and time and treatment effects for P4, E2 and PGFM concentrations. The findings showed that treatment with PGF2α during the periovulatory period has potential to increase fertilization rate and embryo production in superovulated buffaloes.

Different times to perform timed artificial insemination when using a P4/E2/eCG-based protocol in buffalo / Momentos diferentes para executar a inseminação artificial em tempo fixo utilizando o protocolo à base de P4/E2/eCG em búfalas

ABSTRACT: The aim of this study was to evaluate different times for timed artificial insemination (TAI) in buffalo submitted to a P4/E2/eCG-based protocol. In this study, 204 buffaloes were distributed into one of two groups (TAI56, n=103 and TAI64, n=101). At a random stage of the oestrous cycle (Day 0 = D0), in the morning (TAI56, a.m.) or afternoon (TAI64, p.m.), buffaloes received an intravaginal progesterone device (P4; 1.0 g) plus EB (2.0 mg i.m.). On D9 a.m. (TAI56) or p.m. (TAI64), the P4 was removed and buffaloes received PGF2a (0.53 mg i.m. sodium cloprostenol) and eCG (400 IU i.m.). On D10 a.m. (TAI56) or p.m. (TAI64), 24 h after P4 removal, buffaloes were treated with EB (1.0 mg i.m.). Buffaloes from TAI56 and TAI64 were inseminated 56 and 64 h after P4 removal (D11, p.m. and D12, a.m., respectively). Ultrasound examinations were performed on D0 to ascertain ovarian follicular status, at TAI to measure the diameter of the dominant follicle (DF) and D42 for pregnancy diagnosis. The statistical analysis was performed using the GLIMMIX procedure of SAS®. There was no difference between TAI56 and TAI64 for the diameter of the DF at TAI and the pregnancy per TAI. It was concluded that TAI 56 or 64 h after P4 removal did not affect fertility in buffaloes submitted to the induction of ovulation with EB. The present research supports that is possible to perform TAI at any time throughout the day in buffalo synchronized during the non-breeding season.

RESUMO: O objetivo deste estudo foi avaliar diferentes momentos para a realização da IATF em búfalas submetidas a um protocolo à base de P4/E2/eCG. Neste estudo, 204 búfalas foram distribuídas em um de dois grupos (IATF56, n=103 e IATF64, n=101). Em estágio aleatório do ciclo estral (Dia 0 = D0), pela manhã (IATF56, manhã) ou pela tarde (IATF64, tarde), as búfalas receberam um dispositivo intravaginal de progesterona (P4; 1,0 g) e BE (2,0 mg i.m.). No D9 pela manhã (IATF56) ou pela tarde (IATF64), a P4 foi removida e as búfalas receberam PGF2a (0,53 mg i.m. cloprostenol sódico) e eCG (400 UI i.m.). No D10 pela manhã (IATF56) ou pela tarde (IATF64), 24 h após a remoção da P4, as búfalas foram tratadas com BE (1,0 mg i.m.). As búfalas dos grupos IATF56 e IATF64 foram inseminadas 56 e 64 h após a remoção da P4 (D11, pela tarde e D12, pela manhã, respectivamente). Avaliações ultrassonográficas foram realizadas no D0 para verificar o status folicular ovariano, na IATF para medir o diâmetro do folículo dominante (FD) e no D42 para o diagnóstico de gestação. A análise estatística foi realizada utilizando o procedimento GLIMMIX do SAS®. Não houve diferença entre os grupos IATF56 e IATF64 no diâmetro do FD na IATF e na prenhez por IATF. Conclui-se que a IATF 56 ou 64 h após a remoção da P4 não afeta a fertilidade de búfalas submetidas à indução da ovulação com BE. A presente pesquisa evidencia que é possível realizar a IATF durante todo o dia em búfalas sincronizadas durante a estação reprodutiva desfavorável.

Superstimulation prior to the ovum pick-up improves the in vitro embryo production in nulliparous, primiparous and multiparous buffalo (Bubalus bubalis) donors.

The aim of this study was to evaluate the ovarian follicular population, the oocyte yield and the in vitro embryo production (IVEP) of nulliparous (NU), primiparous (PR) and multiparous (MU) buffalo donors submitted to the superstimulation with FSH prior to the ovum pick-up (OPU). A total of 54 buffalo donors (18 NU, 15 PR and 21MU) received an intravaginal progesterone device (1.0 g) plus estradiol benzoate [2.0 mg, intramuscular (im)] at random stage of the estrous cycle (Day 0) during the breeding season (autumn and winter). Buffaloes from different categories were then randomly allocated to one of two groups (Control or FSH), in a cross-over experimental design. Buffalo donors in the Control group received no further treatment, whereas buffalo donors in the FSH group received a total dosage of 200 mg im of FSH on Days 4 and 5, in four decreasing doses 12 h apart (57, 57, 43 and 43 mg). On Day 7, the progesterone device was removed and the OPU procedure was performed in both groups. The same semen was used across all replicates and donor category. Data were analyzed by the GLIMMIX procedure of SAS 9.4®. There was no interaction between FSH treatment and animal category for all analyzed variables. Furthermore, no differences between animal category (P = 0.73) and FSH treatment (P = 0.53) were observed regarding the total follicles aspirated. However, the FSH treatment increased (P < 0.001) the proportion of large (>10 mm; FSH = 16.2% and Control = 2.0%) and medium-sized follicles (6-10 mm; FSH = 36.3% and Control = 6.1%) available for the OPU procedure. The total of recovered oocytes was greater in NU than in MU, and PR were similar to NU and MU (P = 0.05). No effect of FSH treatment was observed (P = 0.85) for this variable. Buffalo donors treated with FSH had a greater viable oocytes rate (P = 0.03), blastocyst rate (P = 0.03) and embryo yield per OPU-IVEP session (P = 0.07), however, no category effects were observed for these variables. These results provided evidence that superstimulation with FSH increased the proportion of large and medium-sized follicles available for the OPU procedure. Consequently, the FSH treatment enhanced the proportion of viable oocytes for culture and resulted in greater blastocyst rates and embryo yield per OPU-IVEP session in all buffalo donors categories.

Challenges to increase the AI and ET markets in Brazil.

Artificial insemination (AI) and embryo transfer (ET) are the most widely used biotechnologies in the world with the goal of increasing genetic gain and improving reproductive efficiency of beef and dairy herds. The protocols for ovulation synchronization for timed AI (TAI) or ET (TET) are tools that allow artificial insemination or transfer of a high number of embryos in a pre-established moment and without the necessity of estrous detection. Currently, 86% of inseminations in Brazil are performed using TAI (13.6 million TAI out of a total of 15.4 million doses of semen marketed in 2018). With the use of TAI, it was possible to verify that the percentage of artificially inseminated females in Brazil went from 5.8% in 2002 to 13.1% in 2018. The ET market also presented considerable growth in the last 20 years. There was an increase of approximately 8 fold in the number of produced embryos, escalating from 50,000 in 1999 to 375,000 in 2017. In this period, there was a significant increase on the in vitro embryo production, which represented 92.1% of embryos produced in Brazil in 2017. Also, in this period, there was an increase on the embryo production of dairy breeds and reduction on the embryo production of zebu breeds in comparison to data from the early 2000's. TET increases significantly the number of recipients suitable to receive an embryo. After synchronization, 75 to 85% of recipients present a suitable CL for ET without estrous detection. Currently, many synchronization and resynchronization protocols for TAI/TET have been studied to attend different managements, breeds and animal categories, with predictable and satisfactory results. With the intensification of the use of these biotechnologies, it is possible to obtain elevated reproductive efficiency with increase on the genetic gain, which determines greater productivity and economic return for dairy and beef farms. However, the challenge to keep the market growing in the next decade could depend on some factors, such as: increase of the extension services for producers and of the extension training for specialists, improvement of the technological advances to develop more efficient and cost-effective products and practical protocols, increase the integration between universities, research institutes, veterinarians and industries and also, asses market demand for production of animal protein with higher quality, efficiency and environmental and economic sustainability.

Effect of season on dairy buffalo reproductive performance when using P4/E2/eCG-based fixed-time artificial insemination management.

This study aimed to compare the reproductive efficiency of dairy buffaloes subjected to TAI protocols based on progesterone, estrogen, and equine chorionic gonadotrophin (P4/E2+eCG) during the fall/winter (n = 168) and spring/summer (n = 183). Buffaloes received an intravaginal P4 device (1.0 g) plus estradiol benzoate (EB; 2.0 mg im) at a random stage of the estrous cycle (D-12). Nine days later (D-3), the P4 device was removed and buffaloes were given PGF2α (0.53 mg im sodium cloprostenol) plus eCG (400 IU im). GnRH (10 µg im buserelin acetate) was administered 48 h after P4 device removal (D-1). All animals were subjected to TAI 16 h after GnRH administration (D0). Frozen-thawed semen from one bull was used for all TAI, which were all performed by the same technician. Ultrasound examinations were performed on D-12 and D-3 to ascertain cyclicity (presence of CL), D-3 and D0 to measure the diameter of the dominant follicle (ØDF), D+10 to verify the ovulation rate and diameter of the corpus luteum (ØCL), and D+30 and D+45 to detect pregnancy rate (P/AI 30d and 45d, respectively) and embryonic mortality (EM). Fetal mortality (FM) was established between 45 days and birth, and pregnancy loss between 30 days and birth. There were significant differences between fall/winter and spring/summer only for cyclicity rate [76.2% (128/168) vs. 42.6% (78/183); P = 0.02]. The others variables did not differ between the seasons: ØDF on D-3 (9.6 ± 0.2 mm vs. 9.8 ± 0.2 mm; P = 0.35); ØDF on D0 (13.1 ± 0.2 mm vs. 13.2 ± 0.2 mm; P = 0.47); ovulation rate [86.9% (146/168) vs. 82.9% (152/182); P = 0.19]; ØCL on D+10 (19.0 ± 0.3 mm vs. 18.4 ± 0.3 mm, P = 0.20); P/AI on D+30 [66.7% (112/168) vs. 62.7% (111/177); P = 0.31]; P/AI on D+45 [64.8%% (107/165) vs. 60.2% (106/176); P = 0.37]; EM [1.8% (2/111) vs. 3.6% (4/110); P = 0.95]; FM [21.9% (18/82) vs. 8.0% (7/87); P = 0.13]; and PL [23.8% (20/84) vs. 12.1% (11/91); P = 0.13]. In conclusion, dairy buffaloes present similar reproductive efficiency in fall/winter and spring/summer when subjected to P4/E2/eCG-based protocol for TAI.