Selecting oocyte donors based on anti-Müllerian hormone (AMH) concentrations: A critical analysis of using cutoff values as exclusion criterion for an in vitro embryo production program in Gir cattle.

The aims of this study were to determine anti-müllerian hormone (AMH) cutoff values for selecting Gir (Bos taurus indicus) oocyte donors and estimate the impact of using AMH concentrations as a selection criterion. In Exp. 1, Gir heifers (n=120) were sampled for AMH analysis and submitted to ovum pick-up and in vitro embryo production (OPU-IVEP). AMH cutoff values were calculated using ROC analysis or, alternatively, by the successive exclusion of heifers with the lowest AMH values. The correlations between AMH and OPU-IVEP outcomes were significant (P<0.001), though low or moderate (r= 0.34-0.52). We estimated an improvement (P<0.05) after the use of AMH cutoff values to select donors of +15.3% for total oocyes, +19.4% for viable COC, and +23.4% for blastocysts. This selection pressure, however, led to the exclusion of 32.8%, 37.9%, and 50.0% of the initial potential donors, respectively. In Exp. 2, we analyzed data from OPU-IVEP sessions of 658 Gir donors with known genomic values for predicted transmitting ability for milk (GPTAm) and age at first calving (GPTAafc). The selection based on the number of oocytes recovered had no effect (P>0.05) on the average GPTAm nor GPTAafc values of the remaining donors. In summary, plasma AMH ≥700 pg/mL is a cutoff value that can be used to select Gir heifers with a greater potential as oocyte donors. Nevertheless, this selection leads to the exclusion of up to 50% of potential donors. Finally, exclusion of poor responders had no effect on mean genomic estimates for milk production or age at first calving in the selected subset of donors.

Effect of maternal age on ATP content and distribution of mitochondria in bovine oocytes.

Our objective was to understand how maternal age influences the mitochondrial population and ATP content of in vivo matured bovine oocytes. We hypothesized that in vivo matured oocytes from older cows would have altered mitochondrial number and distribution patterns and lower cytoplasmic ATP content compared to the oocytes obtained from younger cows. Follicles ≥5mm were ablated in old cows (13 to 22 yrs, Old Group, n = 7) and their younger daughters (4 to 10 years old, Young Group; n = 7) to induce the emergence of a new follicular wave. Cows were treated twice daily with eight doses of FSH starting 24 hr after ablation (Day 0, day of wave emergence). Prostaglandin F2alpha (PGF) was given on Days 3 and 3.5, LH on Day 4.5, and cumulus-oocyte-complexes were collected 18-20 hours post-LH by ultrasound-guided follicular aspiration. Oocytes were either processed for staining with MitoTracker Deep Red FM or for ATP assay. Stained oocytes were imaged with a Zeiss LSM 710 confocal microscope, and mitochondria were segmented in the oocyte volume sets using Imaris Pro 7.4. In vivo matured oocytes obtained from old cows were similar in morphological grades to those from young cows. However, the oocytes of COC from older cows had 23% less intracellular ATP (27.4±1.9 vs 35.7±2.2 pmol per oocyte, P = 0.01) than those of young cows. Furthermore, the average volume of individual mitochondria, indicated by the number of image voxels, was greater (P<0.05) in oocytes from older cows than in those from younger cows. Oocytes from older cows also tended to have a greater number of mitochondrial clusters (P = 0.06) and an increased number of clusters in the central region of the oocytes (P = 0.04) compared to those from younger cows. In conclusion, our study demonstrated that maternal age was associated with a decrease in the cytoplasmic ATP content of in vivo mature oocytes and an altered distribution of mitochondrial structures. These findings suggest that maternal age may negatively influence the developmental competence of oocytes from older cows.

Does GnRH treatment at TAI regardless of estrus occurrence increase pregnancy rate in crossbred Bos taurus suckled cows?

The impact of GnRH treatment on the day of TAI in beef cows has received limited investigation, especially concerning its association with estrus expression. Consequently, two experiments were conducted to assess the potential of GnRH treatment on the day of TAI to enhance fertility according to the expression or not of estrus in beef cows. Experiment 1 aimed to determine ovulation rate and luteal function, while Experiment 2 aimed to determine the effect of the two GnRH treatment approaches on pregnancy rate. In Experiment 1, multiparous Brangus suckling cows (n = 17) were submitted to an 8-day TAI protocol. Estrus occurrence was evaluated based on chalk removal on D10 (TAI) and cows were assigned to receive GnRH (25µg lecirelin; im) according to the group: GnRH (n = 7), regardless of estrus expression; or selectGnRH (n = 10), only cows not detected in estrus. Ovulation rate occurring until 77h after IVD removal did not differ (p = 0.17) between GnRH (85.7%; 6/7) and selectGnRH (100%; 10/10). Also, corpus luteum size and serum progesterone concentration were not affected (p>0.05) by treatments. In Experiment 2, crossbred taurine suckled cows (n = 384) were submitted to the same protocol as described in Experiment 1 and were randomly allocated to GnRH or selectGnRH groups. There was no difference in P/AI between groups (selectGnRH = 55.6%; GnRH = 54.3%; p = 0.7) 30 days after TAI. As expected, there was a pronounced effect (p<0.0001) of estrus expression on P/AI (Estrus = 61.5%; No estrus = 33.0%), regardless of group. In summary, ovulation timing and rate and luteal function did not differ between groups. Also, GnRH administration only in cows that do not show estrus is recommended, considering hormone savings and similar conception rate.

Exploring uterine inflammation in postpartum primiparous precocious and conventional and multiparous Bos indicus beef cows.

This study aimed to compare the postpartum uterine dynamics of primiparous precocious (PP), primiparous conventional (PC) and multiparous conventional (MC) Bos indicus beef cows. For this purpose, PP (n = 8), PC (n = 18) and MC (n = 12) cows were enrolled in this study. These cows were evaluated at 20 and 10 days prepartum and weekly from parturition to 42 days postpartum (DPP). During this period, body weight (BW), subcutaneous fat thickness (SFT) and serum concentrations of glucose, ß-hydroxybutyrate, albumin and haptoglobin were measured. Proportion of polymorphonuclear (PMN) cells, and abundance of mRNA transcripts of genes involved in uterine inflammation and uterine health were evaluated. The PP cows had lower (p < .05) BW and SFT than that for PC and MC cows during the study period. The serum concentration of albumin after 35 DPP was lower (p < .05) in PP cows. The PP cows had the highest proportion of PMN on 28 and 35 DPP compared to PC and MC cows. The relative mRNA abundance of IL-1ß and IL-8 increased after 21 DPP in PP cows compared to the other groups. The PC had the highest, MC had an intermediate, and PP cows had the lowest relative abundance of IL10 mRNA. Overall, these findings indicated that uterine inflammation was more pronounced in PP cows. Moreover, based on the proportion of PMN and abundance of transcripts associated with inflammation in the uterus, PP cows may require a longer period to recover their uterine health after calving.

GnRH34 with or without estradiol cypionate in timed AI in Bos indicus beef cows.

Two experiments were performed to evaluate the effects of GnRH treatment on the fertility of suckled Nelore beef cows treated with an estradiol/progesterone (E2/P4)-based protocol for timed artificial insemination (TAI). Experiment 1 focused on determining the effects of estradiol cypionate (EC) on ovulation in TAI cows treated with GnRH 34 h after removal of the intravaginal P4 device (IPD). Suckled cows (n = 26) were treated with 2 mg estradiol benzoate (EB) and IPD containing 1 g P4. After 8 days, IPDs were removed, and all cows were treated with 150 µg of d-cloprostenol (prostaglandin F2 alpha analog) and 300 IU of equine chorionic gonadotropin (eCG), then separated into two treatment groups consisting of cows who received 1) saline 0.9% i.m. (GnRH34 group) or 2) 0.6 mg i.m. of EC (EC-GnRH34 group). On day 9 (05:00 p.m.), all cows were given GnRH (10.5 µg of buserelin acetate) i.m. No differences were observed between the groups (P > 0.05) in the time of ovulation after IPD removal or in the proportion of cows ovulating. Experiment 2 focused on determining the effects of GnRH34 along with or in the absence of EC on day 8 on pregnancy per AI (P/AI) in postpartum beef cows. Cows (n = 981) were treated similarly to those in Experiment 1, but an additional group, the EC-GnRH48 group, was included, in which cows received EC on day 8 whereas those that did not show estrus received GnRH at TAI. Thus, in this experiment, groups consisted of GnRH34 (n = 322), EC-GnRH34 (n = 335), and EC-GnRH48 (n = 324). A higher rate of estrus expression was observed in cows treated with EC following IPD removal (EC-GnRH34: 69%, EC-GnRH48: 64.8%) than in cows in the GnRH34 group (45.6%). No difference in P/AI was observed between the treatment groups (P = 0.45), but P/AI in cows in the EC-GnRH34 group (64.2%) tended to be greater (P = 0.1) than in cows in the GnRH34 group (58%). In summary, although ovulation synchrony did not differ among the groups, P/AI in cows treated with EC and GnRH 34 h after IPD removal tended to be greater than in cows treated solely with GnRH; this was most likely due to a shorter proestrus/estrus period, considering the lower proportion of cows that displayed estrus in the GnRH34 group. Finally, given that P/AI did not differ between the EC-GnRH34 and EC-GnRH48 groups, our results suggest that, for cows not displaying estrus, administration of EC at the time of IPD removal followed by treatment with GnRH 48 h afterward represents the most cost-efficient TAI strategy for South American Zebu-based beef operations.

Early versus late body condition score loss in dairy cows: Reproductive performance.

This study aimed to compare milk production and reproductive performance in high yield Holstein cows that lose BCS early and late in the postpartum period. Lactating dairy cows (n = 76) were received first timed AI at 60 to 75 DIM using the farm-managed estradiol-progesterone-GnRH-based timed AI protocol. The BCS of all cows was daily evaluated by automated BCS cameras. Aiming to evaluate the effect of the days in milk (DIM) in which a cow reached the nadir BCS on the reproductive parameters, cows were separated into two groups: early BCS loss (n = 42), cows that reached the nadir BCS ≤ 34 DIM, and late BCS loss (n = 34), cows that reached the nadir BCS > 34 DIM. The optimal cut-off point for determining the relationship between days to nadir BCS and pregnancy by 150 DIM (P150) was calculated using the receiver operating characteristic (ROC) curve. From the ROC analysis, the cut-off was 34 DIM (Se, 80.9%; Sp, 66.7%; AUC, 0.74; P < 0.01). No differences (P>0.05) were detected between groups on the BCS and milk production. The average of milk production in both groups was 46.65 ± 6.15 Kg/day. Cows that reached the nadir BCS early postpartum presented lower (P < 0.01) calving interval and greater (P < 0.01) pregnancy at first AI and P150. In summary, cows that lost BCS early had better reproductive performance and had similar milk yield compared with cows that lost BCS late in the postpartum period.

Does combining estradiol cypionate and GnRH for ovulation induction in recipient cows increase pregnancy rate after timed embryo transfer?

Estradiol cypionate (EC) or GnRH have been widely used for ovulation induction in timed embryo transfer (TET). EC administration increases the proportion of cows that show estrus, whereas GnRH promotes more synchronized ovulations. The aim of the present study was to evaluate the potential beneficial effects of combining EC and GnRH in TET. In experiment 1, no difference was observed on serum progesterone concentrations on Day 6 and 13 after GnRH treatment between GnRH and EC+GnRH groups. In experiment 2, pregnancy per embryo transfer (P/ET) did not differ (p = 0.69) between GnRH (62.8%) and EC+GnRH (58.7%) groups. In conclusion, combining EC and GnRH for ovulation induction does not increase progesterone secretion and pregnancy rate after TET in cattle.

Effect of calving to timed artificial insemination interval on fertility of beef cows.

The objectives of this study were to determine the effect of calving to timed artificial insemination (C-TAI) interval on fertility of Bos taurus and Bos indicus cows and to determine the best C-TAI interval to include postpartum cows in TAI programs in each genotype (B. taurus and B. indicus). In experiment 1, a retrospective study was performed, with data from 2,709 TAI in Bos taurus and Bos indicus cows with different C-TAI intervals. There was a positive linear effect of C-TAI interval on the pregnancy probability (p < .0001). However, there was no effect or interaction with the genotype. When C-TAI intervals were grouped in classes, pregnancy rate differed in groups (p < .05). Receiver operating characteristic (ROC) analysis was performed, and it was observed that 52 days would be the better C-TAI interval to maximize fertility in beef cows. According to the previous result, we further stratified into two C-TAI interval groups (≤ 52 days or > 52 days). Cows with C-TAI longer than 52 days (C-TAI > 52) had greater pregnancy rate (56%) than cows with short C-TAI (46%; p < .01). In experiment 2, 18 postpartum beef cows were subjected to TAI protocol at different C-TAI intervals (n = 6 cows per group): 40, 60 or 80 days postpartum. Cows with 60 days of C-TAI had a greater preovulatory follicle diameter than cows with 80 days postpartum (p < .05). The C-TAI interval did not affect (p > .05) the ovulation rate nor the proportion of endometrial polymorphonuclear leucocytes. We conclude that C-TAI interval affects pregnancy rate in both Bos taurus and Bos indicus. We recommend a C-TAI interval longer than 52 days to increase fertility of beef cows included in TAI programs.

GnRH34: An alternative for increasing pregnancy in timed AI beef cows.

Two experiments evaluated the effects of gonadotropin releasing hormone (GnRH) treatment on fertility of suckled Nelore beef cows treated with an estradiol/progesterone (P4)-based protocol for timed artificial insemination (TAI). Experiment 1 was designed to determine the effect of GnRH administered 34 h after P4 insert removal (GnRH34) on time of ovulation. Suckled cows (n = 34) were treated with 2 mg estradiol benzoate (EB) and an intravaginal insert containing 1.9 g of P4. Eight days later, P4 inserts were removed, and all cows received 150 µg of d-cloprostenol (prostaglandin F2 alpha analogue), 300 IU of eCG, and 1 mg of estradiol cypionate (ECP). On Day 9 (05:00 p.m.), cows were randomly distributed, according to the diameter of the pre-ovulatory follicle, in two treatments: 1) GnRH (n = 17) cows that received 10.5 µg of buserelin acetate, or 2) no further treatment (control, n = 17). Cows treated with GnRH 34 h after P4 insert removal ovulated earlier (P = 0.02) than control cows (66 ± 0.0 and 77.2 ± 4.3 h). Experiment 2 was designed to determine the effect of GnRH34 on the fertility of suckled beef cows. Nelore cows (n = 506) were treated as Experiment 1. On Day 8, cows were painted in the sacrocaudal region to identify cows that displayed estrus. On Day 9 (05:00 p.m.), cows were randomized to receive same treatment as Experiment 1, control (n = 252), or GnRH (n = 254). All cows were TAI 48 h after P4 insert removal. At TAI, estrus was evaluated, and deemed to have occurred in cows without a tail-head chalk mark (>75% paint loss). Cows treated with GnRH 34 h after P4 insert removal had greater (P = 0.01) pregnancy per AI (P/AI) than cows that only received ECP (63.0% and 50.4%). No difference (P = 0.5) was observed in the proportion of cows that displayed estrus between treatments. Furthermore, cows that displayed estrus had greater (P < 0.01) P/AI than cows that did not. Treatment with GnRH, given at 34 h after P4 insert removal, increased (P < 0.05) P/AI in cows that did not show estrus at TAI. In summary, treatment with GnRH 34 h after P4 insert removal was associated with earlier ovulation and resulted in greater P/AI in suckled Nelore cows treated with an estradiol/P4-based protocol for TAI.

Prostaglandin F2α regulation and function during ovulation and luteinization in cows.

Although prostaglandins are important in the ovulation process, a precise role for prostaglandin F2α (PGF) has not been elucidated. This study aimed to evaluate the regulation of PGF receptor mRNA (PTGFR) in granulosa cells and the local effect of PGF on ovulation and luteinization. In Experiment 1, using samples collected in vivo before (Day 2), during (Day 3) and after (Day 4) follicular deviation, expression of PTGFR in bovine granulosa cells was more abundant in the dominant follicle after deviation than in subordinates (P < 0.05). However, the expression of PTGFR was not regulated (P = 0.1) in preovulatory follicles at different time-points (0, 3, 6, 12 and 24 h) after ovulation induction with GnRH. In Experiment 2, to assess the role of systemic PGF treatment on luteinization and vascularization of preovulatory follicles, flunixin meglumine (FM), a nonsteroidal anti-inflammatory drug, was used to inhibit endogenous prostaglandin synthesis. Cows with preovulatory follicles were induced to ovulate with GnRH (0 h) and allocated to three groups: Control, with no further treatment; FM, treated with 2.2 mg/kg FM im 17 h after GnRH treatment; and FM + PGF, treated with FM 17 h after GnRH, followed by 25 mg dinoprost tromethamine (PGF) 23 h after GnRH treatment. FM injection was able to reduce the concentration of PGF in the follicular fluid (FF) (P < 0.001). However, contrary to our hypothesis, color Doppler ultrasound evaluations revealed decreased vascular flow in FM + PGF group (P < 0.05), and no effect of the treatments on intrafollicular P4 and E2 concentrations 24 h after GnRH. The prostaglandin metabolite (PGFM) concentrations in the FF were greater in cows receiving systemic PGF (P < 0.001), which prompted us to further check its role on ovulation. Therefore, in Experiment 3, in a final attempt to demonstrate the local effect of PGF on ovulation, cows with preovulatory follicles received an intrafollicular injection (IFI) of PBS (Control) or 100 ng/mL purified PGF (PGF group). PGF treatment did not affect the time of ovulation after IFI (66 ± 6.4 and 63 ± 8.5 h for control and PGF, respectively; P > 0.05), further suggesting that it has no direct effect in the ovulatory process. Based on our findings, we concluded that FM decreased PGF synthesis within the follicle, whereas PGF treatment decreased follicular vascularization. In addition, the in vivo model of intrafollicular injection evidenced that PGF alone is not able to locally induce ovulation.

The role of prostaglandin F2α on ovulation and LH release in cows / A função da prostaglandina F2α na ovulação e na liberação de LH em vacas

This study aimed to evaluate the role of prostaglandin F2α (PGF) on ovulation. In Experiment 1, cows were randomly allocated to two treatments to receive 150 µg of d-Cloprostenol (PGF Group, n = 12) or 2 mL of NaCl 0.9% (Control Group, n = 11) and CIDRs, were removed 4 days later. No cow ovulated in Control and PGF groups. In Experiment 2, cows were randomly separated into two experimental groups to receive 4 injections of 150 µg of d-Cloprostenol (n = 9) or 2 mL of NaCL 0.9% (n = 9). In this experiment, ovulation was not observed in any cows. In Experiment 3, ovariectomized cows receive three injections of 300µg of PGF analog (PGF Group, n = 5), 100µg of Lecirelin (GnRH Group, n = 5) or 2 mL of PBS (Control Group, n = 4). The LH concentration was higher (P <0.0001) in cows from the GnRH group than in the PGF and Control groups. In experiment 4, cows with preovulatory follicles (>11.5 mm) were treated with Saline (Control Group, n = 6); Lecirelin (GnRH Group, n = 7) or Cloprostenol Sodium (PGF Group, n = 6). There was a significant increase in the vascular area of follicles from 0 to 24 h in GnRH and PGF treatments. In conclusion, PGF was not able to induce ovulation in cows with high or low plasma progesterone concentration. Additionally, PGF alone was not able to induce LH release and follicle luteinization, but increased follicular vascularization.(AU)

O objetivo deste estudo foi avaliar o papel da prostaglandina F2α (PGF) na ovulação. No Experimento 1, as vacas foram alocadas aleatoriamente em dois tratamentos para receber 150 µg de d-Cloprostenol (Grupo PGF, n = 12) ou 2 mL de NaCl 0,9% (Grupo Controle, n = 11) e os CIDR, foram removidos 4 dias depois. Nenhuma vaca ovulou nos grupos Controle e PGF. No Experimento 2, as vacas foram separadas aleatoriamente em dois grupos experimentais para receber 4 injeções de 150 µg de d-Cloprostenol (n = 9) ou 2 mL de NaCL 0,9% (n = 9). Não foi observada ovulação em nenhum dos animais deste experimento. No Experimento 3, vacas ovariectomizadas receberam três injeções de 300µg de análogo de PGF (Grupo PGF, n = 5), 100µg de Lecirelina (Grupo GnRH, n = 5) ou 2 mL de PBS (Grupo Controle, n = 4). A concentração de LH foi maior (P <0,0001) nas vacas do grupo GnRH do que nos grupos PGF e Controle. No Experimento 4, vacas com folículos pré-ovulatórios (> 11,5 mm) foram tratadas com solução salina (Grupo Controle, n = 6), Lecirelina (Grupo GnRH, n = 7) ou Cloprostenol Sódico (Grupo PGF, n = 6). Houve um aumento significativo na área vascular dos folículos de 0 a 24h nos tratamentos com GnRH e PGF. Em conclusão, a PGF não foi capaz de induzir ovulação em vacas com alta ou baixa concentração plasmática de progesterona. Além disso, a PGF sozinha não foi capaz de induzir a liberação de LH e a luteinização do folículo, mas aumentou a vascularização folicular.(AU)

Prostaglandin E2 induces ovulation in prepubertal mice / Prostaglandina E2 induz ovulação em camundongos pré-púberes

The objective of this study was to determine the ability of prostaglandin E2 (PGE2) to induce ovulation and expression of PGE2 receptor (EP2 and EP4) and COX genes (COX-1 and COX-2) in the ovary and pituitary of prepubertal mice. The positive control consisted of the application of 5 µg of gonadotropin-releasing hormone (GnRH, n = 29); the negative control applied 0.5 mL of phosphate buffered saline (PBS, n=31); the treatment tested the application of 250 µg of PGE2 (n = 29), making a total of 89 prepubertal mice (BALB/c). Mice were euthanized 14 to 15 h after treatments to detect ovulation and tissue collection. A Chi-square test was used to compare the proportion of animals ovulating. Gene expressions and number of ovulation were analyzed by one-way ANOVA and Tukey's test was used to compare means among groups. A greater proportion of mice (P < 0.001) ovulated after receiving GnRH (89.7%, 26/29) compared to PGE2 group (58.6%, 17/29). However, the proportion was higher compared to those treated with PBS (0%, 0/31). Ep2gene expression in the pituitary was > two-fold higher (P < 0.05) in the PGE2 group compared to the PBS and GnRH groups. Further, PGE2 stimulated Cox1 (2.7 fold, P < 0.05) while GnRH stimulated Cox2 expression (6.5 fold, P < 0.05) in the pituitary when compared to the PBS group. In conclusion, our results support the hypothesis that PGE2 can induce ovulation in prepubertal mice with a concomitant increase in Ep2 and Cox1 gene expression in the pituitary gland.(AU)

O objetivo deste estudo foi determinar a capacidade da prostaglandina E2 (PGE2) em induzir a ovulação e expressão do receptor PGE2 (EP2 e EP4) e genes COX (COX-1 e COX-2) no ovário e na hipófise de camundongos pré-púberes. O controle positivo consistiu na aplicação de 5 µg de hormônio liberador de gonadotrofina (GnRH, n = 29); o controle negativo aplicação 0,5 mL de tampão fosfato-salino (PBS, n=31); o tratamento testado aplicação de 250 µg de PGE2 (n = 29), perfazendo um total de 89 camundongos (BALB/c) pré-púberes. Os camundongos foram sacrificados 14 a 15 h após os tratamentos para detectar ovulações e coleta de tecido. O teste do qui-quadrado foi usado para comparar a proporção de animais ovulando. As expressões gênicas e o número de ovulação foram analisados por ANOVA e o teste de tukey foi usado para comparar as médias entre os grupos. Uma maior proporção de camundongos (P <0,001) ovulou após receber GnRH (89,7%, 26/29) em comparação com o grupo PGE2 (58,6%, 17/29). No entanto, a proporção foi maior em comparação com aqueles tratados com PBS (0%, 0/31). A expressão do gene Ep2 na hipófise foi duas vezes maior (P <0,05) no grupo PGE2 em comparação com os grupos PBS e GnRH. Além disso, a PGE2 estimulou a Cox1(2,7 vezes, P <0,05) enquanto o GnRH estimulou a expressão de Cox2 (6,5 vezes, P <0,05) na pituitária em comparação com o grupo PBS. Em conclusão, nossos resultados suportam a hipótese de que PGE2 é capaz de induzir ovulação em camundongos pré-púberes com aumento concomitante na expressão dos genes Ep2 e Cox1 na glândula pituitária.(AU)

Effects of Prostaglandins E2 and F2α on the in vitro maturation of bovine oocytes.

We aimed to elucidate the effects of PGE2 and PGF2α on the in vitro maturation (IVM) of bovine oocytes. First, cumulus-oocyte complexes were matured in the media supplemented with or without PGE2, PGF2α, or PGE2 plus PGF2α for the final 24, 12, or 6 h of culture. Then, the cumulus-oocyte complexes were matured in the absence or presence of a PG endoperoxide synthase 2 (PTGS2) enzyme inhibitor (NS398) supplemented with PGE2, PGF2α, or PGE2 plus PGF2α. Finally, the expression of genes associated with PGs activity in cumulus cells (PTGS2, PG E-synthase-1 [PTGES1], and aldo-keto reductase 1 [AKR1B1]) or oocytes (receptors for PGE2 [PTGER2] and PGF2α [PTGFR]) of different competencies was quantified. Supplementation of the IVM medium with PGs did not improve in vitro embryo production or embryo quality (P > 0.05). During maturation, the relative abundance of PTGS2 transcripts increased (P < 0.05) only in the less-competent group, whereas those of PTGES1 increased in the less-competent and in the more-competent groups. Conversely, AKR1B1 expression decreased only in the less-competent group (P < 0.05). Receptors for the PGE2 and PGF2α genes were very low or undetectable in oocytes. In conclusion, PGE2 and PGF2α are not recommended for media supplementation during maturation because they have no effect on embryo development. Although genes related to PGs activity are differentially expressed in cumulus cells of cumulus-oocyte complexes of different competence during maturation, the expression of PGE2 and PGF2α receptor genes was either not detectable or was detected at low levels in oocytes.

Intrafollicular lipopolysaccharide injection delays ovulation in cows.

The aim of this study was to evaluate effects of intra-follicular (i.f.) treatment with lipopolysaccharide (LPS) on follicular and luteal development in cows. There were 18 non-lactating cows assigned to two groups to address this aim: control group (n = 9), which received an i.f. injection of saline; and LPS group (n = 9), which received an i.f. injection of 1 µg of LPS per mL of follicular fluid. Cows were treated with an intravaginal P4 releasing device (IVD) and estradiol benzoate on D0. On D4 and D5 cows were treated with cloprostenol sodium and on D7 the IVD was removed. At 12 h after IVD removal, cows were administered the i.f. injection of LPS or saline. After administration of these treatments, follicular development was evaluated every 12 h until ovulation. The LPS treatment increased blood flow in pre-ovulatory follicles (P = 0.05). Follicle growth was reduced by LPS injection (P < 0.02) resulting a longer period to the time of ovulation for cows in the LPS than control group (P = 0.03). The percentage of cows having ovulations was less for the LPS than control group (P = 0.03). The diameter of the CL, CL blood flow and P4 concentrations 5 and 12 days after ovulation did not differ between groups (P> 0.05). In conclusion, intra-follicular treatment with LPS resulted in a decreased rate of follicle growth, delayed timing of ovulations and a lesser number of cows having ovulations.

Vascular and morphological features of the corpus luteum 12 to 20 days after timed artificial insemination in dairy cattle.

Our objective was to retrospectively compare pregnant versus nonpregnant cattle in terms of vascular and morphometric changes in corpora lutea between d 12 and 20 following timed artificial insemination (TAI). Crossbred (Gir × Holstein) lactating dairy cows (n = 136) and heifers (n = 111) were bred after synchronizing ovulations using an estradiol plus progesterone (P4)-based protocol. Corpus luteum (CL) characteristics (area, echotexture, blood flow) were recorded at 48-h intervals from d 12 to 20 following TAI using an ultrasound equipped with color Doppler. Blood samples were collected to determine CL function (plasma P4). Pregnancy diagnosis was performed at d 30. Quantitative assessment of colored pixels within the CL was performed using ImageJ software (National Institutes of Health, Bethesda, MD) and echotexture was quantified using custom software. Continuous variables such as luteal tissue area (LTA), CL blood flow (CLBF), adjusted CLBF (ratio LTA:CLBF), mean pixel value (MPV), pixel heterogeneity (HETER), and plasma P4 were analyzed retrospectively as repeated measures (d 12 to 20) in pregnant versus nonpregnant females using PROC MIXED (SAS Institute Inc., Cary, NC). Main effects were pregnancy status, day of cycle, and their interaction. Further analyses used only data from d 16, because this was the earliest time point of deviation between CLBF of pregnant and nonpregnant animals. We created quartiles for each variable and calculated the risk of pregnancy within quartile. Differences were determined using the chi-squared test. Plasma P4 was significantly higher in prospective pregnant versus nonpregnant cattle on d 18 and 20, whereas LTA differed only on d 20. On d 16, CLBF and adjusted CLBF diverged between pregnant and nonpregnant, followed by a progressive reduction in the latter until d 20. Mean pixel value was not affected by pregnancy status, but HETER was lower on d 20 in pregnant than in nonpregnant cattle. Likelihood of pregnancy increased from quartile (Q)1 (lowest values) to Q4 (highest) of CLBF (Q4 vs. Q1, odds ratio = 32.8, 95% confidence interval: 9.6 to 112.1) and adjusted CLBF [Q4 vs. Q1, odds ratio = 25.4, 95% confidence interval: 8.1 to 80.4), whereas a lower risk of pregnancy was observed only for animals within Q1 of plasma P4 [Q4 vs. Q1, odds ratio = 3.1, 95% confidence interval: 1.3 to 7.2). Day 16 quartiles of LTA, MPV, and HETER did not affect odds of pregnancy. In conclusion, we identified distinct CLBF patterns as early as 16 d after TAI and confirmed that CL function is lost by a reduction in blood flow, which precedes physical regression.

Effect of sperm kinematics and size of follicle at ovulation on pregnancy rate after timed AI of beef cows.

The objectives of this study were to evaluate whether the diameter of the preovulatory follicle and the post-thaw sperm kinematics are factors that affect successful establishment of a pregnancy after timed AI (TAI) of postpartum beef cows. Nelore cows (n = 346) were subjected to an estradiol-progesterone based TAI protocol. At TAI, cows were categorized according to the diameter of the preovulatory follicle (POF) in the Early Ovulation (EO group, n = 184), and Late Ovulation (LO group, n = 162) groups. Sperm were classified, according to CASA analysis, as Hyper-activated (H+), and Non-Hyper-activated (H-). The H + and H- semen was used to inseminate cows that were classified to be in the EO and LO groups. Thus, after distribution, the groups were: EOH+ (n = 89), EOH- (n = 95), LOH+ (n = 96), and LOH- (n = 66). Pregnancy per AI (P/AI) was greater (P < 0.05) for EOH+ (66.3%, 59/89), EOH- (65.3%, 62/95), and LOH- (72.7%, 48/66) groups than for LOH + group (52.1%, 50/96). The results of the study indicate that cows with a smaller POF are less likely to become pregnant when insemination is with semen considered as hyper-activated (H+). In contrast, when inseminations are performed with semen categorized to be non-hyper-activated (H-), cows of the EO and LO groups had acceptable and similar pregnancy rates as a result of timed AI.

Uterine inflammation and fertility of beef cows subjected to timed AI at different days postpartum.

The aim of this study was to evaluate the uterine health and fertility of postpartum beef cows subjected to timed AI (TAI) protocols at different days from calving. Lactating Nelore cows (Bos indicus; n = 244) were used in this study. The TAI protocols were initiated between 20 and 60 days postpartum (DPP). Cows were subjected to an estradiol-progesterone based TAI protocol. The cows were divided into three groups according to the days postpartum at the time the hormonal treatment was initiated: 1) Early (n = 64), cows between 20 and 30 DPP; 2) Middle (n = 115), cows between 31 and 45 DPP; and 3) Late (n = 65), cows between 46 and 60 DPP. At Day 0 of the protocol, endometrial cytobrush samples were collected. Slides for polymorphonuclear (PMN) cell counting were prepared and cytobrush was used for RNA extraction and analysis of relative abundance of il1, il6, il8, and tnf mRNA. Cows from the Early group had less (P < 0.05) pregnancies per AI (P/AI) than cows from Middle and Late groups; 29.7% (19/64), 45.2% (52/115), and 52.31% (34/65), respectively. Accordingly, the Early group had a greater (P < 0.05) proportion of PMN cells in the uterus than Middle and Late groups; 9.0%, 3.8%, and 2.2%, respectively. Relative abundances of il1 and il8 mRNA were greater (P < 0.05) in the Early group than Middle and Late groups. These results indicate that beef cows subjected to TAI protocols early postpartum (< 30 DPP) are less likely to become pregnant, which is associated with increased inflammation and relative abundance of mRNA for the proinflammatory cytokines il1 and il8.

Different protocols using PGF2α as ovulation inducer in Nelore cows subjected to estradiol-progesterone timed AI based protocols.

The objective of this study was to evaluate the effect of a PGF2α-analogue (PGF) on ovulation and pregnancy rates after timed artificial insemination (TAI) in cattle. In Experiment 1 cows received an intravaginal progesterone-releasing device (CIDR) plus 2 mg im of estradiol benzoate (EB) on Day 0. The CIDR devices were removed on Day 8, and all cows received 150 µg im of d-cloprostenol (PGF2α-analogue), 300 IU of eCG and 1 mg of estradiol cypionate (ECP) im. On Day 9, cows were randomly assigned into two groups: 1) ECP Group (n = 17), that did not receive any further treatment; and 2) ECP-PG Group (n = 14) that were given 150 µg of d-cloprostenol (PGF) as adjuvant stimulus for ovulation. No difference between groups was detected in interval for ovulation (P = 0.5), and in the proportion of cows ovulating (P = 0.09). In Experiment 2, multiparous suckling crossbred Aberdeen Angus cows (n = 260), were treated into two groups, similarly as Experiment 1; ECP group (n = 122), and ECP-PG group (n = 138). All females were TAI on Day 10. The proportion of cows treated with ECP that became pregnant was 54.9% (67/122), and cows treated with ECP plus PGF was 55.1% (76/138; P = 0.9). In Experiment 3, 686 Nelore cows, 40 to 50 days postpartum, were treated as Experiment 1 (ECP group), however, on Day 8 cows were divided into 3 groups: ECP Group (n = 216); PGF-SC Group (n = 228), in which cows did not receive ECP and were given an additional subcutaneous injection of PGF on Day 8; and PGF-IM Group (n = 242), in which cows also did not receive ECP on Day 8 and were given an additional injection of PGF im on Day 9. On Day 10, estrus was evaluated at timed AI (TAI). There was no difference in the diameter of the dominant follicle at CIDR removal and at TAI, and pregnancy per AI among groups (P > 0.05). However, the proportion of cows that displayed estrus between CIDR removal and TAI was higher in ECP group than in PGF-SC and PGF-IM groups (P < 0.001). Cows that displayed estrus has higher P/AI than cows that did not (P = 0.008). In conclusion, these results suggested that intramuscular or subcutaneous injection of PGF2α could be successfully used to induce ovulation in cattle undergoing TAI, with similar pregnancy rates when compared with ECP. The subcutaneous injection of PGF on the same day of CIDR removal could be an interesting alternative due it reduces cattle management to obtain similar results.

Effect of injectable progesterone on follicular development in lactating beef cows treated with estradiol plus a low-concentration progesterone device / Efeito da progesterona injetável no desenvolvimento folicular em vacas de corte lactantes tratadas com estradiol e dispositivo de progesterona de baixa concentração

The effect of injectable progesterone was evaluated along with estradiol benzoate (EB) on the fate of the dominant follicle (DF) present in the ovary at the beginning of low progesterone-based TAI protocol. All cattle were given 500 µg cloprostenol im (PGF; Schering-Plough Animal Health for Estrumate, Pointe-Claire, QC, Canada) twice, 11 d apart, and allocated into two groups: Estradiol group (E group, n = 11) and Estradiol-Progesterone group (EP group, n = 11). Ten days after the second PGF (Day 0), all cattle were given an intravaginal progesterone device with half progesterone concentration (Cue-Mate with a single pod containing 0.78 g progesterone). Concurrently, all cattle were given 1.5 mg im of estradiol benzoate in 3 mL of canola oil and PGF im on Day 0 of the protocol in a crossover design, in which each cow received both treatments. Cows in the EP group also received 100 mg im progesterone (Sigma) in 2 mL of canola oil. On Day 8, progesterone devices were removed and all cattle were given PGF im. All statistical analyses were performed with SAS 9.0. The DF present on Day 0 ovulated in 76% (16/21) of cows from E group and 28.6% (6/21) of cows from EP group (P = 0.002). After progesterone device removal, the size of ovulatory follicle did not differ between groups (E group, 15.5 ± 0.43 mm vs EP group, 15.8 ± 0.98 mm; P = 0.82). These follicles ovulated in 81.3 ± 3.1 h in E group and 71.0 ± 6.1 h in EP group (P = 0.13). In conclusion, injectable progesterone reduced the proportion of cows that ovulate the dominant follicle present in the ovary at the beginning of estradiol-progesterone-based protocols. However, no difference was detected on time of ovulation after progesterone device removal between groups.(AU)

Foi avaliado o efeito da progesterona injetável e do benzoato de estradiol (BE) no destino do olículo dominante (FD) presente no ovário no início do protocolo de IATF. Todas as vacas receberam duas injeções de 500 µg de cloprostenol im (PGF; Schering-Plough Animal Health for Estrumate, Pointe-Claire, QC, Canadá) em um intervalo de onze dias e foram alocadas em dois grupos: Estradiol (grupo E, n = 11) e Estradiol-Progesterona (grupo EP, n = 11). Dez dias após a segunda injeção de PGF (Dia 0), elas receberam um implante intravaginal de progesterona com metade da concentração hormonal (Cue-Mate com apenas uma haste contendo 0,78 g de progesterona). Além disso, todas vacas receberam 1,5 mg im de BE dissolvido em óleo de canola e PGF im no Dia 0 do protocolo, em um delineamento em crossover no qual cada vaca recebeu ambos tratamentos. Vacas do grupo EP ainda receberam uma injeção de 100 mg im de progesterona (Sigma) em 2 mL de óleo de canola no Dia 0. No Dia 8, os dispositivos de progesterona foram removidos e todas as vacas receberam PGF im. A análise estatística foi realizada por meio do pacote estatístico SAS 9.0. O FD presente no Dia 0 ovulou em 76% (16/21) das vacas do grupo E e em 28,6% (6/21) das vacas do grupo EP (P = 0,002). Após a remoção do dispositivo de progesterona, o diâmetro do folículo ovulatório não apresentou qualquer diferença entre os grupos (grupo E, 15,5 ± 0,43 mm; grupo EP, 15,8 ± 0,98 mm; P = 0,82). Esses folículos ovularam em 81,3 ± 3,1 h no grupo E e em 71,0 ± 6,1 h no grupo EP (P = 0,13). A conclusão obtida foi que o uso de progesterona injetável reduziu a proporção de vacas que ovularam o folículo dominante presente no ovário no início do protocolo à base de estradiol e progesterona. No entanto, entre os grupos não houve diferença no momento da ovulação após a remoção do dispositivo de progesterona.(AU)

Effect of serum paraoxonase-1 (PON1) activity on follicular development and pregnancy rate in cattle.

Paraoxonase-1 (PON1) activity has been associated with improvement in ovarian function in early postpartum dairy cows and improved in vitro embryo development. The aim of the current study was to evaluate the potential association among PON1 activity and follicular growth, diameter of the preovulatory follicle and pregnancy per artificial insemination (AI) service in cattle. In Experiment 1, cows (n=33) were subjected to an estradiol-progesterone based protocol to control time of ovulation. Starting on Day 8 of the protocol, follicular growth and serum PON1 activity were monitored. Cows were separated according to the occurrence of ovulation into two groups: Ovulatory (Ov; n=22) and Anovulatory (Anov; n=11). The serum activity of PON1 was not different between Ov and Anov cows (P=0.94). In addition, using a regression model there was no effect of serum PON1 activity on the diameter of dominant follicle (r2=0.00; P=0.99). In Experiment 2, cows (n=193) were submitted to the same hormonal protocol as in Experiment 1. On the day of the timed artificial insemination (TAI), the diameter of dominant follicle was evaluated and blood samples were collected for analysis of PON1 activity. According to the serum PON1 activity, cows were divided into three groups: Low (<70U/mL), Medium (70-90U/mL) or High (>90U/mL) PON1 activity. The overall pregnancy rate was 62.7% (121/193), with no difference among PON1 activity groups. Additionally, using a regression model there was no effect of serum PON1 activity on the diameter of the preovulatory follicle (r2=0.03; P=0.65) and pregnancy rate (r2=0.005; P=0.94). The results of this study indicate that there is no effect of serum PON1 activity on the diameter of preovulatory follicle or establishment of pregnancy in cows submitted to time of ovulation synchronization protocols.