Gene expression profile in heat-shocked Holstein and Nelore oocytes and cumulus cells.

The present study determined the transcriptome profile in Nelore and Holstein oocytes subjected to heat shock during IVM and the mRNA abundance of selected candidate genes in Nelore and Holstein heat-shocked oocytes and cumulus cells (CC). Holstein and Nelore cows were subjected to in vivo follicle aspiration. Cumulus-oocyte complexes were assigned to control (38.5°C, 22h) or heat shock (41°C for 12h, followed by 38.5°C for 10h) treatment during IVM. Denuded oocytes were subjected to bovine microarray analysis. Transcriptome analysis demonstrated 127, nine and six genes were differentially expressed between breed, temperature and the breed×temperature interaction respectively. Selected differentially expressed genes were evaluated by real-time polymerase chain reaction in oocytes and respective CC. The molecular motor kinesin family member 3A (KIF3A) was upregulated in Holstein oocytes, whereas the pro-apoptotic gene death-associated protein (DAP) and the membrane trafficking gene DENN/MADD domain containing 3 (DENND3) were downregulated in Holstein oocytes. Nelore CC showed increased transcript abundance for tight junction claudin 11 (CLDN11), whereas Holstein CC showed increased transcript abundance for antioxidant metallothionein 1E (MT1E) . Moreover, heat shock downregulated antioxidant MT1E mRNA expression in CC. In conclusion, oocyte transcriptome analysis indicated a strong difference between breeds involving organisation and cell death. In CC, both breed and temperature affected mRNA abundance, involving cellular organisation and oxidative stress.

Progesterone-based fixed-time artificial insemination protocols for dairy cows: Gonadotropin-releasing hormone versus estradiol benzoate at initiation and estradiol cypionate versus estradiol benzoate at the end.

Our objectives were to evaluate ovarian dynamics and fertility comparing 2 treatments at the start of a progesterone (P4)-based fixed-time artificial insemination (FTAI) protocol and 2 treatments at the end of the protocol. Thus, 1,035 lactating Holstein cows were assigned in a random phase of the estrous cycle to 1 of 4 treatments using a completely randomized design with a 2×2 factorial arrangement. At the beginning of the protocol (d -10), cows received GnRH or estradiol benzoate (EB) and, at the end, EB (d -1) or estradiol cypionate (ECP; d -2), resulting in 4 treatments: GnRH-EB, GnRH-ECP, EB-EB, and EB-ECP. All cows received an intravaginal P4 device on d -10, which was removed on d -2. Cows also received PGF2α on d -3 and -2. The FTAI was performed on d 0. Ovaries were evaluated by ultrasound for corpus luteum (CL) presence and regression (d -10 and -3) and follicle measurements (d -10 and 0), as well as the uterus for percentage pregnant per AI (P/AI; d 32 and 60). Blood samples were collected (d -10 and -3) for P4 measurements. Treatment with GnRH rather than EB tended to increase P/AI on d 32 (38.2 vs. 33.7%) and on d 60 (32.9 vs. 28.9%). More cows treated with GnRH had CL on d -3 compared with EB-treated cows (77.3 vs. 58.3%), due to less CL regression between d -10 and -3 (24.7 vs. 43.8%) and more cows with a new CL on d -3 (35.9 vs. 25.0%). Cows treated with GnRH also had greater P4 concentrations on d -3 than EB cows (3.4 vs. 2.0 ng/mL). Increased circulating P4 at the start of the protocol (d -10) decreased the probability of ovulation to EB or GnRH at that time. Cows from GnRH group also ovulated a larger-diameter follicle at the end of the protocol (15.5 vs. 14.7mm). No difference between EB and ECP in P/AI on d 32 (34.8 vs. 37.0) and 60 (30.8 vs. 31.0%) or in pregnancy loss (11.1 vs. 15.4%) was detected and we found no interaction between treatments for P/AI. Independent of treatment, cows with CL on d -10 and -3 had the greatest P/AI on d 60 (36.9%). In conclusion, treatments at the end of the protocol were similar for ECP or EB and we found no additive effect or interactions on P/AI between treatments. However, cows treated with GnRH rather than EB on d -10 had less luteolysis and tended to have greater P/AI, probably because P4 concentrations were greater during the protocol. Finally, regardless of treatments, cows with CL at the beginning of the protocol as well as at the time of PGF2α had greater fertility.

Progesterone supplementation to lactating dairy cows without a corpus luteum at initiation of the Ovsynch protocol.

The objectives were to determine the effect of progesterone supplementation on fertility responses in lactating dairy cows without corpora lutea (CL) at initiation of the timed artificial insemination (AI) program. Holstein cows from 5 commercial dairy farms were subjected to the Ovsynch-56 protocol (d -10 GnRH, d -3 PGF2α, d -0.7 GnRH, d 0 AI). Ovaries were scanned by ultrasonography on d -10. Within farm, cows without CL were blocked by pen and assigned randomly to remain as nonsupplemented controls (CON; n = 652) or to receive 2 controlled internal drug-release (CIDR) inserts containing 1.38 g of progesterone each from d -10 to -3 (2CIDR; n = 642). Cows with CL were randomly selected within pen and used as positive controls as cows in diestrus at the initiation of the Ovsynch protocol (DIEST; n = 640). Signs of estrus were detected beginning on d -9 based on removal of tail chalk, and cows in estrus received AI on the same day. Blood samples from subsets of cows on d -10, -9, -7, -5, -3, and 0 (n = 109) and on d 6, 13, and 19 (n = 156) were analyzed for progesterone concentrations. Pregnancy was diagnosed on d 32 and 60 after AI. The average progesterone concentration during the timed AI program was lowest for CON, intermediate for 2CIDR, and highest for DIEST (0.92, 2.77, and 4.93 ng/mL, respectively). The proportions of cows that ovulated in response to the first GnRH (63.6, 61.1, and 47.2%, respectively) and that had a new CL on d -3 at PGF2α injection (72.4, 67.9, and 47.4%, respectively) were greater for CON and 2CIDR compared with DIEST, respectively. The diameter of the ovulatory follicle and the proportion of cows that ovulated in response to the second GnRH did not differ among treatments. A greater proportion of CON and 2CIDR cows were detected in estrus at AI compared with DIEST cows (35.8, 39.6, and 30.6%, respectively). Pregnancy per AI was less for CON compared with 2CIDR and DIEST on d 32 (31.3, 42.2, and 38.4%, respectively) and d 60 after AI (28.9, 37.2, and 33.9%, respectively), indicating that progesterone supplementation reestablished fertility in cows lacking a CL similar to that of cows in diestrus at the initiation of the timed AI program. Treatment did not affect pregnancy loss between d 32 and 60 of gestation. Pregnancy from a subset of cows with plasma progesterone concentrations indicated that a minimum concentration of 2.0 ng/mL was needed to optimize fertility. A single ultrasound examination effectively identified a low-fertility cohort of cows based on the absence of CL at the first GnRH injection of the Ovsynch protocol. Supplementation with 2 CIDR inserts increased progesterone in plasma by an additional 1.85 ng/mL compared with CON, resulting in concentrations of 2.77 ng/mL during development of the ovulatory follicle, which restored fertility in dairy cows lacking CL to a level similar to that of cows in diestrus.