The main objectives of the present study were to determine the ultrastructural modifications occurring in the oocyte during late folliculogenesis and to estimate pre-antral follicle population in buffalo. Half the collected ovaries were fixed and prepared for optic microscopy; the antral follicles from the other ovaries were measured and individually punctured. The cumulus-oocyte complexes (COCs) were processed for transmission electron microscopy. The number of pre-antral follicles in buffalo ovaries was estimated at 19 819 structures. Cumulus-oocyte complexes derived from 1-mm antral follicle had an eccentrical nucleus and compact corona radiata, ooplasm vilosities were fully embedded in zona pellucida (ZP) and a well-defined junction could be observed. Mitochondria were predominantly round and well distributed in ooplasm, as were small lipid vacuoles. In COCs derived from 2-mm antral follicles, the initial formation of perivitelline space was observed. The nucleus was peripherally located and the number of pleomorphic mitochondria increased. Cortical granules were clustered at oocyte periphery and lipid vacuoles increased in number and size. In COCs derived from 6-mm antral follicles, the organelles were located mainly in the perinuclear region. Golgi complexes and smooth endoplasmic reticulum (SER) were more developed. Mitochondria migrated to the cortical region and lipid vacuoles migrated to the medullar region. In COCs derived from 10-mm antral follicles, the lipid vacuoles coalesced and occupied the medullar region of the oocyte, together with a well-developed SER. Mitochondria were pleomorphic and located at the oocyte periphery. In conclusion, the morphological differences described in this paper could be responsible for some functional differences observed in in vitro embryo production and follicular dynamics for buffalo, when compared with cattle.

This study evaluates the post-hatching development of in vitro-produced (IVP) embryos until Day 14. On Day 7, IVP embryos were either transferred to recipient uteruses or placed in a post-hatching development (PHD) system. As a control group, in vivo-produced (IVV), Day-7 embryos were also transferred to recipient uteruses. All groups were collected on Day 14 and were morphologically evaluated. Day-7 and Day-14 IVV and IVP embryos were used for quantification of eight genes (PLAC8, CD9, SLC2A1, SLC2A3, KRT8, SOD2, HSP1A1, and IFNT2) by reverse transcriptase qPCR. Day-14 embryos from the PHD system were smaller (2.92 ± 0.45 mm) and had a lower embryonic disk diameter (0.14 ± 0.00 mm) than those produced by IVV (24.18 ± 3.71; 0.29 ± 0.03 mm, respectively) or IVP (19.06 ± 2.43; 0.28 ± 0.01 mm) culture and transferred to the uterus (P > 0.05). Day-7 IVP embryos had a higher expression of the HSP1A1, SCL2A1, and SCL2A3 genes than IVV embryos. When these embryos were cultured in the uterus, no differences in gene expression were observed on Day 14. Conversely, Day-14 IVP embryos cultured in the PHD system showed a higher expression of PLAC8, SOD2, and SLC2A3 genes. It is concluded that Day-7 IVP embryos are different from IVV embryos in regards to gene expression, although exposure to the uterine environment during the elongation period allowed the IVP embryos to overcome this difference. In contrast, IVP embryos cultured in the PHD system were morphologically and molecularly different, being of poorer quality than those cultured in the uterus.

The objective of the present study was to describe ultrastructural changes in the nucleus and cytoplasmic organelles during in vitro maturation (IVM) of buffalo cumulus-oocyte complexes (COCs). The structures were collected by ovum pick-up (OPU). Some COCs, removed from maturation medium at 0, 6, 12, 18 and 24 h, were processed for transmission electron microscopy. The average number of COCs collected by OPU/animal/session was 6.4, and 44% of them were viable. Immature oocytes had a peripherally located nucleus, Golgi complex and mitochondrial clusters, as well as a large number of coalescent lipid vacuoles. After 6 h of IVM, the oocyte nucleus morphology changed from round to a flatter shape, and the granulosa cells (GC) lost most of their contact with zona pellucida (ZP). At 12 h the first polar body was extruded and the aspect of lipid droplet changed to dark, probably denoting lipid oxidation. Cortical granules were clearly visible at 18 h of maturation, always located along the oocyte periphery. At 24 h of IVM the number of cortical granules increased. Ultrastructure studies revealed that: (1) immature oocytes have a high lipid content; (2) the perivitelline space (PS) increases during IVM; (3) Golgi complexes and mitochondrial clusters migrate to oocyte periphery during IVM; (4) 6 h of IVM are enough to lose contact between GC and ZP; (5) the oocyte lipid droplets' appearance changes between 6 and 12 h of IVM.

The present study aimed to identify biomarkers to assess the quality of in vitro produced (IVP) bovine embryos in the culture media. IVP embryos on Day (D) 5 of development were transferred to individual drops, where they were maintained for the last 48 h of culture. Thereafter, the medium was collected and the embryos were transferred to the recipients. After pregnancy diagnosis, the media were grouped into the pregnant and nonpregnant groups. The metabolic profiles of the media were analyzed via electrospray ionization mass spectrometry, and the concentrations of pyruvate, lactate, and glutamate were assessed using fluorimetry. The spectrometric profile revealed that the media from embryos from the pregnant group presented a higher signal intensity compared to that of the nonpregnant group; the ions 156.13 Da [M + H]+, 444.33 Da [M + H]+, and 305.97 Da [M + H]+ were identified as biomarkers. Spent culture medium from expanded blastocysts (Bx) that established pregnancy had a greater concentration of pyruvate (p = 0.0174) and lesser concentration of lactate (p = 0.042) than spent culture medium from Bx that did not establish pregnancy. Moreover, pyruvate in the culture media of Bx can predict pregnancy with 90.9% sensitivity and 75% specificity. In conclusion, we identified markers in the culture media that helped in assessing the most viable IVP embryos with a greater potential to establish pregnancy.

This study evaluated the effects of three maturation systems, namely invitro (MatV) and invivo (MatS) systems, as well as intrafollicular transfer of immature oocytes (IFIOT; MatT), on the accumulation of lipid droplets in bovine oocytes. Lipids were evaluated using confocal microscopy and transmission electron microscopy. The expression of genes related to lipid metabolism, namely acyl-CoA synthetase short chain family member 2 (ACSS2), ELOVL fatty acid elongase 1 (ELOVL1) and fatty acid binding protein 3 (FABP3), was quantified by quantitative polymerase chain reaction. The mean (±s.d.) area occupied by lipids in immature oocytes (13±2%) was similar to those matured invivo (MatS, 16±2%; MatT, 12±2%). However, there was a significant increase in lipids in oocytes in the MatV group (24±2%) compared with all other groups (P<0.001). In the ultrastructural evaluations, MatV oocytes also showed the highest lipid content. The expression of ELOVL1 and FABP3 was similar in the MatS and IFIOT groups. However, transcript levels of ACSS2 were lower in IFIOT than MatV oocytes. These results indicate, for the first time, that oocytes matured by IFIOT are similar to those matured invivo with regard to lipid accumulation, which indicates better quality than those matured invitro.

Objetivou-se avaliar a eficácia da metil-formamida na criopreservação do sêmen ovino. O pool de sêmen utilizado no experimento foi obtido a partir da coleta, com vagina artificial, do sêmen de quatro carneiros mestiços Santa Inês, com idade aproximada de quatro anos. As coletas foram realizadas uma vez por semana, por seis semanas consecutivas, correspondendo, cada semana, a uma repetição do experimento. As frações do pool foram diluídas em cinco diferentes meios de congelação: (1) trisgema com 5,3% de glicerol (TG5,3G); (2) tris-gema com 3% de metil-formamida (TG3MF); (3) tris-gema com 5% de metilformamida (TG5MF); (4) tris-gema com 7% de metil-formamida (TG7MF); (5) tris-gema com 9% de metil-formamida (TG9MF). Foram avaliadas a motilidade progressiva e o vigor das células espermáticas e realizado o teste de termorresistência pós-descongelação. O tratamento que obteve maior motilidade foi o TG5,3G (50%), seguido do TG3MF (38%) e os tratamentos que apresentaram menor motilidade progressiva foram TG5MF (29%), TG7MF (1,0%), TG9MF (6,0%). Os meios contendo metil-formamida apresentaram resultados inferiores ao meio controle para preservar a integridade morfológica dos espermatozoides, sendo que nos meios TG7MF e TG9MF menos de 60% de espermatozóides apresentaram-se morfologicamente normais. Os espermatozoides do meio TG5,3G apresentaram motilidade (15%) e vigor (2,8) similares aos do meio TG3MF (15% e 2,6, respectivamente) no teste de termorresistência, mas o meio TG5,3G preservou melhor a integridade funcional da membrana plasmática. O glicerol foi mais eficiente como crioprotetor do que a metilformamida na criopreservação de sêmen ovino.

Cows intensively used as oocyte donors for in vitro embryo production (IVEP) are usually kept nonpregnant for prolonged intervals, exposed to successive hormonal treatments, and frequently become overweight. These are all risk factors for the development of endocrine unbalance and, consequently, cystic ovarian disease (COD). The aim of this study was to evaluate the effect of active immunization against gonadotropin-releasing hormone (GnRH) on (1) ovarian follicular population, and (2) development potential of oocytes used for IVEP. Nelore (Bos indicus) cows (n = 14), previously diagnosed with chronic COD (Faria et al. 2017 Anim. Reprod., in press), weighing 620.0 ± 12.8 kg and with body condition score of 4.1 ± 0.2, were assigned to control (n = 6) or treatment (n = 8) group. Cows in the treatment group received 2 SC injections of 1.0 mL of anti-GnRH vaccine (Bopriva, Zoetis, Brazil), 28 days apart (weeks 0 and 4), whereas cows in the control group received placebo on the same schedule. Transrectal ultrasonography was performed weekly from week 0 to evaluate the number and distribution of follicles among size classes, endometrial thickness, and clinical presence of mucometra. Immunization was considered effective (E-IM) when no follicles ≥5.0 mm were observed on the ovaries during a given examination. Cows having E-IM were then used as oocyte donors for IVEP. Cumulus-oocyte complexes (COC) were collected in 5 consecutive ovum pick-up weekly sessions. As a control for IVEP, oocytes from a slaughterhouse were used, with similar procedures performed on the same days and using the same semen batch. The MIXED procedure of SAS (SAS Institute Inc., Cary, NC, USA) with repeated-measures statement was used to evaluate the effects of treatment, time, and interactions on ovarian endpoints; and the GLM procedure was used to analyse embryo production data. Results are shown as mean ± SEM. There were time and time × treatment effects on ovarian parameters. Treated cows had a decrease (P < 0.05) in the average diameter of the largest follicle and in the number of follicles ≥8 mm, and an increase (P < 0.05) in follicular population after week 6. Nonetheless, individual response to treatment was variable: only 50% of the cows (4 of 8) were E-IM at week 8, whereas 25% (2 of 8) still had COD (largest follicle ≥18.0 mm) at this timepoint. Overall, a negative correlation was detected between follicular population and the diameter of the largest follicle (r = –0.60, P < 0.0001) or the number of follicles ≥8 mm (r = –0.47, P < 0.0001). There was no effect (P > 0.05) of treatment on endometrial thickness or mucometra score. Cows with E-IM produced 22.2 ± 3.6 total and 12.9 ± 2.3 viable COC. Cleavage rate did not differ between E-IM and control (slaughterhouse) oocytes (70.8 ± 7.0 v. 75.1 ± 3.0%, respectively; P > 0.05); however, blastocyst rate was greater in the E-IM group compared with controls (39.7 ± 5.5 v. 20.5 ± 4.7%, respectively; P < 0.02). In summary, our results suggest that active immunization against GnRH leads to variable results in the distribution of the follicular population in cows with COD, but it does not negatively affect IVEP efficiency. This research was supported by Zoetis, CNPq, and CAPES.

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