Influence of sperm post-thaw subpopulations of angus and nelore bulls on pregnancy rates by fixed-time artificial insemination.

BACKGROUND: The heterogeneity of ejaculate indicates that fertility is still variable among bulls and that more stringent evaluation methods are needed to identify the ejaculates suitable for AI. OBJECTIVE: To identify and characterize the sperm subpopulations (SP) in thawed semen doses of Nelore and Angus bulls and to evaluate the influence of these sperm subpopulations on pregnancy rate in cows submitted to fixed-time AI (FTAI). MATERIALS AND METHODS: A dose of post-thawed semen from each bull (n=18; consisting of Angus n = 9 and Nelore n = 9) was analyzed for: sperm kinetics; morphology and plasma membrane integrity; and the determination of the sperm subpopulations. Differences between the groups were estimated with the t-test considering a significance level of <5%. RESULTS: There was no influence between breeding bulls for sperm morphology, plasma membrane integrity, and pregnancy rate (P > 0.05). Regarding the kinetic parameters evaluated by the CASA system, Nelore had greater values, for cells with slow velocity (Angus: 16.4 %; Nelore: 21.7%; P = 0.028). In contrast, ANGUS bulls had more static cells (Angus: 27.2%; Nelore: 9.3%; P = 0.048). Based on CASA system data and clustering procedures, four sperm subpopulations were statistically established. In Angus bulls, a higher level of fast and nonlinear spermatozoa were found in SP3 (33.3%), followed by SP1 (32.7%%) with fast and progressive spermatozoa. Whereas, SP1 of Nelore bulls had 33.8% fast and progressive spermatozoa, followed by 32.2% of SP3 with fast and nonlinear spermatozoa. CONCLUSION: Both breeds of bulls presented similar proportions of sperm SP. Consequently, no influence on the pregnancy rates was shown in cows submitted to the IATF programs on a large scale. doi.org/10.54680/fr22310110312.

Antral follicle population in prepubertal and pubertal heifers.

The antral follicle count (AFC) is an important tool in the selection of bovine females destined for biotechnology. However, little is known about AFC in prepubertal and pubertal heifers. Some challenges inherent to the physiology of young females must be considered to achieve efficient rates with different procedures, such as ovum pick-up and IVF. This paper covers some important topics about ovarian physiology related to the population of antral follicles and reproductive efficiency in young female cattle.

Alpha lipoic acid (ALA) effects on developmental competence of equine preantral follicles in short-term culture.

The effect of different concentrations of alpha lipoic acid (ALA) on the development and morphology of preantral follicles, as well as the proliferative activity of granulosa cells, was assessed after short-term culture. Ovaries (n = 5) of five seasonal anestrous mares were harvested in a local abattoir. At the laboratory, nine ovarian fragments (5 × 5 × 1 mm) from each animal were used. One fragment was immediately fixed in Bouin and subjected to histological and immunohistochemistry (proliferating cell nuclear antigen, PCNA) analyses (noncultured group; D0 = day 0). The other eight fragments were cultured in situ for two (D2) or six (D6) days in MEM+ or MEM+ plus ALA (50, 100, or 250 µM). After culture, fragments were subjected to histology and PCNA analyses. After two days of culture, ALA 50 and ALA 100 had the greatest (P < 0.05) percentage of normal primordial follicles (97.2 and 95.1%, respectively), when compared to other groups, and did not differ (P > 0.05) from the fresh noncultured control group. Furthermore, the total percentage of normal follicles was greater (P < 0.05) in the ALA 50 and ALA 100 than in the MEM-D2 group. After six days of culture, the highest (P < 0.05) proliferative activity of granulosa cells in developing follicles was observed for the groups MEM+ (92.9%), ALA 50 (100%), and ALA 100 (96.4%). In conclusion, the results of this study demonstrated that (1) ALA 50 and ALA 100 preserved the morphological integrity of equine primordial follicles for up two days of culture, and (2) granulosa cells of developing follicles enclosed in ovarian tissue and cultured for up to six days in MEM+ with or without ALA were highly stained by PCNA.

Improvement of development of equine preantral follicles after 6 days of in vitro culture with ascorbic acid supplementation.

The aim of this study was to evaluate the effects of different concentrations of ascorbic acid (25, 50, and 100 µg/mL) in supplemented minimum essential medium (MEM+) on the development of equine preantral follicles that were cultured in vitro for 2 or 6 days. The contralateral ovaries (n = 5) from five mares in seasonal anestrus were collected from a local abattoir. Nine ovarian tissue fragments of approximately 5 × 5 × 1 mm were obtained from each animal. One fragment was immediately fixed and subjected to histologic analysis (control group; Day 0), and the other eight were placed in PBS supplemented with penicillin (200 IU/mL) and streptomycin (200 mg/mL) at 4 °C for 1 hour (during transport to the laboratory). The fragments were cultured in situ for 2 days (D2) or 6 days (D6) in MEM+ or MEM+ plus ascorbic acid at three different concentrations, establishing the following nine groups: control; MEM+ (D2); MEM+ (D6); MEM+ 25 µg/mL of ascorbic acid (D2); MEM+ 25 µg/mL of ascorbic acid (D6); MEM+ 50 µg/mL of ascorbic acid (D2); MEM+ 50 µg/mL of ascorbic acid (D6); MEM+ 100 µg/mL of ascorbic acid (D2); and MEM+ 100 µg/mL of ascorbic acid (D6). The preantral follicles were classified according to their stage (primordial, primary, secondary, or antral) and their morphology (normal or abnormal). Slides (n = 951) including 4450 histologic sections were evaluated. Follicles were observed in only 4.85% (216 of 4450) of the histologic sections. Of the 407 follicles evaluated, 120 were in the primordial stage and 287 were in different developmental stages; additionally, 43.5% were morphologically normal. After 6 days of culture, the groups cultured with 50 and 100 µg/mL of ascorbic acid differed in terms of follicular development compared with the other groups. On the basis of occurrence of follicular development and the presence of viable follicles, it can be concluded that a positive effect of culture for 6 days in MEM+ supplemented with 50 and 100 µg/mL of ascorbic acid was observed on equine ovarian fragments.

Dynamics of follicular growth and progesterone concentrations in cyclic and anestrous suckling Nelore cows (Bos indicus) treated with progesterone, equine chorionic gonadotropin, or temporary calf removal.

The objective of this study was to investigate the effects of eCG and temporary calf removal (TCR) associated with progesterone (P4) treatment on the dynamics of follicular growth, CL size, and P4 concentrations in cyclic (n = 36) and anestrous (n = 30) Nelore cows. Cyclic (C) and anestrous (A) cows were divided into three groups. The control group received 2 mg of estradiol benzoate via intramuscular (IM) injection and an intravaginal device containing 1.9 g of P4 on Day 0. On Day 8, the device was removed, and the animals received 12.5 mg of dinoprost tromethamine IM. After 24 hours, the animals received 1 mg of estradiol benzoate IM. In the eCG group, cows received the same treatment described for the control group but also received 400 UI of eCG at the time of device removal. In the TCR group, calves were separated from the cows for 56 hours after device removal. Ultrasound exams were performed every 24 hours after device removal until the time of ovulation and 12 days after ovulation to measure the size of the CL. On the same day as the CL measurement, blood was collected to determine the plasma P4 level. Statistical analyses were performed with a significance level of P ≤ 0.05. In cyclic cows, the presence of the CL at the beginning of protocol resulted in a smaller follicle diameter at the time of device removal (7.4 ± 0.3 mm in cows with CL vs. 8.9 ± 0.4 mm in cows without CL; P = 0.03). All cows ovulated within 72 hours after device removal. Anestrous cows treated with eCG or TCR showed follicle diameter at fixed-timed artificial insemination (A-eCG 10.2 ± 0.3 and A-TCR 10.3 ± 0.5 mm) and follicular growth rate (A-eCG 1.5 ± 0.2 and A-TCR 1.3 ± 0.1 mm/day) similar to cyclic cows (C-eCG 11.0 ± 0.6 and C-TCR 12.0 ± 0.5 mm) and (C-eCG 1.4 ± 0.2 and C-TCR 1.6 ± 0.2 mm/day, respectively; P ≤ 0.05). Despite the similarities in CL size, the average P4 concentration was higher in the A-TCR (9.6 ± 1.4 ng/mL) than in the A-control (4.0 ± 1.0 ng/mL) and C-TCR (4.4 ± 1.0 ng/mL) groups (P < 0.05). From these results, we conclude that eCG treatment and TCR improved the fertility of anestrous cows by providing follicular growth rates and size of dominant follicles similar to cyclic cows. Additionally, TCR increases the plasma concentrations of P4 in anestrous cows.

Effect of holding medium, temperature and time on structural integrity of equine ovarian follicles during the non-breeding season.

The objective was to evaluate the efficiency of phosphate-buffered saline (PBS) and Minimum Essential Medium (MEM) during the transport of equine preantral and antral follicles at various temperatures and incubation interval. Equine ovaries (n = 10) from an abattoir were cut into 19 fragments; one was immediately fixed in Bouin's solution (control) and the other fragments were placed in PBS or MEM solution at 4, 20, or 39 °C for 4, 12, or 24 h. After the respective incubation periods, all fragments were fixed in Bouin's solution for 24 h and then submitted to standard histologic analysis. In total, 2567 ovarian follicles were analyzed, including 1752 primordial, 764 primary, 34 secondary and seven antral follicles. Relative to the control group, the transport of equine ovarian fragments in both solutions significantly reduced the percentage of morphologically normal follicles with increasing time and temperature. At 4 °C for 4 h, considering primordial and developing follicles, PBS had a higher (P < 0.05) rate (98.9%) of morphologically normal follicles than MEM, 48.7%. At 39 °C for 12 h, all follicles in both solutions were degenerated. Regarding the stage of follicular development, primordial follicles were less (P < 0.05) affected by preservation than primary and secondary follicles in all media, times and temperatures tested, except at 4 °C for 12 h in PBS, in which the primary and secondary follicles were less (P < 0.05) affected. Overall, 43% of antral follicles were morphologically normal when maintained in MEM at 4 °C for 4 h. In conclusion, equine follicles were successfully preserved in ovarian fragments at 4 °C in phosphate-buffered saline for up to 4 h.

Comparison of embryo yield and pregnancy rate between in vivo and in vitro methods in the same Nelore (Bos indicus) donor cows.

To investigate why the preferred means to produce bovine embryos in Brazil has changed from in vivo to in vitro, we compared these two approaches in the same Nelore cows (n=30) and assessed total embryo production and pregnancy rates. Without a specific schedule, all cows were subjected to ultrasound-guided ovum pick up (OPU)/in vitro production (IVP) and MOET, with intervals ranging from 15 to 45 d between procedures, respectively. To produce in vivo embryos, cows were superovulated and embryos were recovered nonsurgically from 1 to 3 times (1.4+/-0.6), whereas OPU/IVP was repeated from 1 to 5 times (3.2+/-1.2) in each donor cow during a 12-mo interval. Embryos obtained from both methods were transferred to crossbred heifers. On average, 25.6+/-15.3 immature oocytes were collected per OPU attempt. The average number of embryos produced by OPU/IVP (9.4+/-5.3) was higher (P<0.05) than the MOET method (6.7+/-3.7). However, pregnancy rates were lower (P<0.05) following transfer of IVP (33.5%) versus in vivo-derived embryos (41.5%) embryos. Embryonic losses between Days 30 and 60 and fetal sex ratio were similar (P>0.05) between in vivo and in vitro-derived embryos. We concluded that in Nelore cows, with an interval of 15 d between OPU procedures, it was possible to produce more embryos and pregnancies compared to conventional MOET.

The nucleotide sequence of Saccharomyces cerevisiae chromosome VII.

The complete nucleotide sequence of Saccharomyces cerevisiae chromosome VII has 572 predicted open reading frames (ORFs), of which 341 are new. No correlation was found between G+C content and gene density along the chromosome, and their variations are random. Of the ORFs, 17% show high similarity to human proteins. Almost half of the ORFs could be classified in functional categories, and there is a slight increase in the number of transcription (7.0%) and translation (5.2%) factors when compared with the complete S. cerevisiae genome. Accurate verification procedures demonstrate that there are less than two errors per 10,000 base pairs in the published sequence.

Sequencing of a 9.9 kb segment on the right arm of yeast chromosome VII reveals four open reading frames, including PFK1, the gene coding for succinyl-CoA synthetase (beta-chain) and two ORFs sharing homology with ORFs of the yeast chromosome VIII.

A 9.9 kb DNA fragment from the right arm of chromosome VII of Saccharomyces cerevisiae has been sequenced and analysed. The sequence contains four open reading frames (ORFs) longer than 100 amino acids. One gene, PFK1, has already been cloned and sequenced and the other one is the probable yeast gene coding for the beta-subunit of the succinyl-CoA synthetase. The two remaining ORFs share homology with the deduced amino acid sequence (and their physical arrangement is similar to that) of the YHR161c and YHR162w ORFs from chromosome VIII.

Sequencing of a 17.6 kb segment on the right arm of yeast chromosome VII reveals 12 ORFs, including CCT, ADE3 and TR-I genes, homologues of the yeast PMT and EF1G genes, of the human and bacterial electron-transferring flavoproteins (beta-chain) and of the Escherichia coli phosphoserine phosphohydrolase, and five new ORFs.

A 17.6 kb DNA fragment from the right arm of chromosome VII of Saccharomyces cerevisiae has been sequenced and analysed. The sequence contains twelve open reading frames (ORFs) longer than 100 amino acids. Three genes had already been cloned and sequenced: CCT, ADE3 and TR-I. Two ORFs are similar to other yeast genes: G7722 with the YAL023 (PMT2) and PMT1 genes, encoding two integral membrane proteins, and G7727 with the first half of the genes encoding elongation factors 1gamma, TEF3 and TEF4. Two other ORFs, G7742 and G7744, are most probably yeast orthologues of the human and Paracoccus denitrificans electron-transferring flavoproteins (beta chain) and of the Escherichia coli phosphoserine phosphohydrolase. The five remaining identified ORFs do not show detectable homology with other protein sequences deposited in data banks. The sequence has been deposited in the EMBL data library under Accession Number Z49133.

The complete sequence of a 9000 bp fragment of the right arm of Saccharomyces cerevisiae chromosome VII contains four previously unknown open reading frames.

We report the sequence of a 9000 bp fragment from the right arm of Saccharomyces cerevisiae chromosome VII. Analysis of the sequence revealed four complete previously unknown open reading frames, which were named G7587, G7589, G7591 and G7594 following standard rules for provisional nomenclature. Outstanding features of some of these proteins were the homology of the putative protein coded by G7589 with proteins involved in transcription regulation and the transmembrane domains predicted in the putative protein coded by G7591.

Analysis of a 17.4 kb DNA segment of yeast chromosome II encompassing the ribosomal protein L19 as well as proteins with homologies to components of the hnRNP and snRNP complexes and to the human proliferation-associated p120 antigen.

We report the nucleotide sequence of a 17.4 kb DNA segment from the left arm of Saccharomyces cerevisiae chromosome II. This sequence contains 12 open reading frames (ORFs) longer than 300 bp and a putative autonomously replicating sequence (ARS). The ORF YBL0418 contains the KH motif present in several nucleic acid-binding proteins and shares homologies with the mouse X protein of the heterogeneous nuclear ribonucleoprotein (hnRNP) complexes involved in pre-mRNA processing. YBL0424 is the yeast member of the ribosomal protein L19 (YL14) family. YBL0425 is related to the D1 core polypeptide of the small nuclear ribonucleoprotein (snRNP) particles involved in the splicing of introns. YBL0437 is a putative homologue of the human protein p120, one of the major antigens associated with malignant tumours. Mcm2, a protein important for ARS activity, as well as Aac2, one of the three isoforms of the mitochondrial ATP/ADP carrier, were previously described (Yan et al., 1991; Lawson and Douglas, 1988). Four ORFs show no homology or particular features that could help to assess their functions. The last ORFs are not likely to be expressed for they are localized on the complementary strand of longer ORFs.