The combined use of timed AI (TAI) and embryo transfer (TET) has the potential to increase reproductive efficiency in beef cattle. This study evaluated reproductive performance in beef cattle after TAI followed by TET of in vitro-produced embryos at the onset of the breeding season. A total of 476 multiparous non-suckling Bos taurus females (body condition scores of 2.9 ± 0.4 on 1 to 5 scale) were oestrous synchronized with 2 mg of oestradiol benzoate (IM) and a 1.9-g intravaginal progesterone release device (Day –11), which was removed on Day –2, followed by 0.48 mg of sodium cloprostenol, 400 IU of eCG, and 0.5 mg of oestradiol cypionate (IM). In experiment I [no heat detection (HD), or no HD, n = 387], TAI was carried out 48 h later (Day 0), whereas in experiment II (after HD, n = 89), AI was performed 12 h after the onset of oestrus up to 48 h after intravaginal insert removal, when remaining females were inseminated (Day 0). Day-7 blastocysts produced by IVF from abattoir-derived oocytes were individually transferred (TET) 7 days after TAI (Day 7) to 186/387 and 44/89 females in experiments I and II, respectively, ipsilateral to the corpus luteum. Then, fertile mature Bos taurus bulls were introduced on Day 12 into the herds (1:25) up to Day 90. Determinations of pregnancy outcome after TAI, TAI+TET or natural mating, twinning rates, and pregnancy losses were done by ultrasonography and rectal palpation on Days 30, 60, and 125. Data were analysed by the Chi-squared test (P < 0.05). Pregnancy rates (Day 30) were lower after TAI (104/201, 51.7%) than after TAI+TET (126/186, 67.7%) with no HD (experiment I), but similar between TAI (32/45, 71.1%) and TAI+TET (30/44, 68.2%) after HD (experiment II). Twinning rates were lower in TAI groups with no HD (6/104, 5.8%) and after HD (2/32, 6.2%) than in TAI+TET groups with either no HD (42/126, 33.3%) or with HD (14/30, 46.7%). Overall pregnancy was similar between groups after the end of the breeding season: 90.0% (181/201) and 90.3% (168/186) for TAI and TAI+TET with no HD, and 84.4% (38/45) and 84.1% (37/44) for TAI and TAI+TET after HD. Pregnancy losses were higher after TAI+TET with no HD (27/126, 21.4%) than TAI+TET after HD (3/30, 10.0%), and TAI with (2/32, 6.3%) or without (9/104, 8.7%) HD. The TAI+TET with no HD resulted in fewer fetuses per served (0.69) and pregnant (1.30) female than TAI+TET after HD (0.89 and 1.44), whereas TAI with no HD had fewer fetuses than TAI after HD per served (0.50 v. 0.69) but not per pregnant female (1.05 v. 1.03), with both being lower than the TAI+TET groups. In summary, TET after TAI with no HD increased pregnancy and twinning rates. Also, heat detection increased pregnancy rates after TAI and twinning rates after TAI+TET. The TAI+TET combination may be advisable for reproductive schemes with no HD, whereas no benefit of TAI+TET was seen over TAI regarding pregnancy rate if TAI is coupled with HD, but HD may increase prolificacy after TAI+TET. The economics of the use of TAI+TET is under evaluation, by assessing calving, weaning, and postnatal weight gain rates between groups.

Cryopreservation of oocytes and embryos is an essential technique for invitro-produced cattle worldwide. One of the great difficulties of cryopreservation of oocytes and blastocysts is the accumulation of lipids in the cytoplasm when produced invitro. The lipid metabolism of oocytes and embryos is classically regulated by the cAMP pathway. Furthermore, previous studies have suggested that the cyclic guanosine monophosphate (cGMP) pathway may also be involved in modulating lipid metabolism through protein kinase G activation. The objective of this study was to investigate the lipid profile of bovine blastocysts produced invitro when stimulated by specific stimulator of cGMP synthesis (NPPB). Pools of oocytes were matured invitro for 24h in tissue culture medium 199, with 15% bovine serum, 0.5µgmL−1 FSH, 5µgmL−1 LH, 0.8mM L-glutamine, and 50µgmL−1 gentamicin at 38.5°C and 10−6 M NPPB. The control group was matured without NPPB. After 22h, the oocytes were fertilised invitro with frozen sperm. The IVM oocytes were fertilised and cultured according standard procedures (Rubessa et al. 2011 Theriogenology 76, 1347-1355). After the 7 days (Day 7), the blastocysts (from early blastocyst to expanded blastocyst) were collected, washed in methanol:water (vol/vol) 1:3, and frozen at −80°C. The lipid extraction of the samples was performed based on the standard protocol (Bligh and Dyer 1959 Can. J. Biochem. Physiol. 37, 911-917) but adapted for small samples. The samples were diluted and analysed in the Agilent 6410 QQQ (Agilent Technologies) mass spectrometer and analysed according to the multiple reaction monitoring method described by de Lima et al. (2018 J. Mass Spectrom. 53, 1247-1252). Data for 3 replicates/group were normalized and then submitted to t-test statistical analysis and principal component analysis, by Metaboanalyst 4.0, with a significance level of 5%. The rates of cleavage and blastocysts were not affected when we used the mGC stimulator presenting a 61% rate of cleavage for both groups, and 24.4% and 25% of blastocyst rate for control and NPPB, respectively (P&lt;0.05). The results, regarding 164 lipids analysed, showed that the lipid profile was not affected when we used NPPB, maintaining the same profile of lipid classes. When we observe the quantitative values, we see a nonsignificant decrease in the lipid classes sphingomyelin, phosphatidylcholine, and triacylglycerol. The values for each class for control and NPPB, respectively, were 0.70 and 0.64 ng/blastocyst for sphingomyelin, 6.45 and 6.07 ng/blastocyst for phosphatidylcholine, and 11.82 and 10.51 ng/blastocyst for triacylglycerol (P&lt;0.05). For the other classes of phospholipids (PE, PG, and PI), we observed a small increase when treated with NPPB, also not significant. We conclude that although we do not have significant differences between the control and the treatment, each class of lipid can respond differently when stimulated with cGMP synthesis.

Intrafollicular lipid metabolism is very important for production species such as cattle. Lipids are essential substrates to produce energy during growth, maturation, and acquisition of high competence for the development of oocytes. However, the quantity and distribution of these lipids has been identified as responsible for hindering the process of cryopreservation of oocytes and embryos produced invitro. Previous studies have indicated that the cyclic (c) GMP pathway may be involved in the lipid metabolism of bovine cumulus-oocyte complexes (COC). The synthesis of this nucleotide can be activated through guanylate cyclases (soluble, sGC; or membrane, mGC). Therefore, the objective of this study was to investigate the lipid profile of bovine oocytes matured invitro (IVM) when stimulated by specific stimulators of sGC (protoporphyrin IX) and mGC (NPPB: peptide natriuretic type B). Pools of ovum pickup (OPU) oocytes were matured invitro for 24h in TCM-199 medium, with 15% bovine serum (BS), 0.5µgmL−1 of FSH, 5µgmL−1 of LH, 0.8mM L-glutamine, and 50µgmL−1 of gentamicin at 38.5°C and 10−5 M protoporphyrin IX or 10−6 M NPPB. The control group was matured without NPs or protoporphyrin IX. After IVM, cumulus cells (CC) were removed and oocytes (OO) collected, washed in 1:3 methanol:water (v/v) and frozen at −80°C. The lipid extraction of the samples was performed based on a standard protocol (Bligh and Dyer 1959 Can. J. Biochem. Physiol. 37, 911-917) but adapted for small samples. The samples were diluted and analysed on an Agilent 6410 QQQ (Agilent Technologies) mass spectrometer and analysed according to the multiple reaction monitoring (MRM) method described (de Lima et al. 2018 J. Mass. Spectrom. 53, 1247-52). Data for 3 replicates/group were normalized and then submitted to ANOVA statistical analysis, followed by Tukey test and principal components analysis, by Metaboanalyst 4.0, with an α-level of 5%. The results, representing the analysis of 164 lipids, showed that the lipid profile was not affected when we used the cGMP synthesis stimulators protoporphyrin IX and NPPB, maintaining the same profile of lipid classes in control and treatments. In addition, the quantitative values of the major lipid classes, sphingomyelin, triglycerides, and phospholipids (phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, phosphatidylglycerol, and phosphatidylinositol), were not altered in the treated groups. The values for each class (ng/oocyte) for control, NPPB, and protoporphyrin, respectively, were 0.89, 0.86, and 1.12 for sphingomyelin, 5.63, 5.66, and 6.90 for phosphatidylcholine, 7.34, 6.48, and 7.89 for triglycerides, 209.0, 244.0, and 207.4 for phosphatidylserine, 3.05, 3.0, and 2.35 for phosphatidylethanolamine, 3.40, 3.34, and 3.29 for phosphatidylglycerol, and 3.47, 3.52, and 3.51 for phosphatidylinositol (P&lt;0.05). Further, the amount of these lipids per class was not affected by cGMP synthesis when stimulated by protoporphyrin IX and NPPB, showing that the relationship of this pathway with lipid metabolism needs additional study.

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