Can in vitro embryo production be estimated from semen variables in Senepol breed by using artificial intelligence?

Thoroughly analyzing the sperm and exploring the information obtained using artificial intelligence (AI) could be the key to improving fertility estimation. Artificial neural networks have already been applied to calculate zootechnical indices in animals and predict fertility in humans. This method of estimating the results of reproductive biotechnologies, such as in vitro embryo production (IVEP) in cattle, could be valuable for livestock production. This study was developed to model IVEP estimates in Senepol animals based on various sperm attributes, through retrospective data from 290 IVEP routines performed using 38 commercial doses of semen from Senepol bulls. All sperm samples that had undergone the same procedure during sperm selection for in vitro fertilization were evaluated using a computer-assisted sperm analysis (CASA) system to define sperm subpopulations. Sperm morphology was also analyzed in a wet preparation, and the integrity of the plasma and acrosomal membranes, mitochondrial potential, oxidative status, and chromatin resistance were evaluated using flow cytometry. A previous study identified three sperm subpopulations in such samples and the information used in tandem with other sperm quality variables to perform an AI analysis. AI analysis generated models that estimated IVEP based on the season, donor, percentage of viable oocytes, and 18 other sperm predictor variables. The accuracy of the results obtained for the three best AI models for predicting the IVEP was 90.7, 75.3, and 79.6%, respectively. Therefore, applying this AI technique would enable the estimation of high or low embryo production for individual bulls based on the sperm analysis information.

Evaluation of cooling and freezing systems of bovine semen.

Semen cryopreservation comprises different steps, among them are the cooling and freezing rates which significantly influence the quality of thawed sperm. Different systems with variable freezing rates are used for freezing bull semen in the field, with a consequence of variable success rates. The objective of this study was to compare different systems for freezing bull semen in the field. Five cooling methods of semen and two methods for the subsequent freezing phase (5 × 2 factorial scheme) were used. Two to four ejaculates were collected from 12 bulls with an electroejaculator. The ejaculates were diluted in BotuBov® to a concentration of 50 × 106 spermatozoa/mL in 0.5-mL straws. After dilution, the straws were cooled to 5 °C in five cooling systems: TK 4000® at a cooling rate of -0.25 °C/min (R1); TK 4000® at a rate of -0.5 °C/min (R2); Minitube® refrigerator at a rate of -2.8 °C/min (R3); Botutainer® at a rate of -0.65 °C (R4), and domestic refrigerator at a rate of -2.0 °C/min (R5). After stabilization at 5 °C for 4 h, these straws were then submitted to two freezing systems: TK 4000® at a freezing rate of -15 °C/min (C1) and Styrofoam box with liquid nitrogen at a rate of -19 °C/min (C2). Sperm kinetics were evaluated by computer-assisted sperm analysis at four time points: in fresh semen, after cooling, post-thawing, and after the rapid thermal resistance test (TRT). In addition, plasma and acrosomal membrane integrity, mitochondrial potential and intracellular H2O2 were analyzed after thawing by flow cytometry. The R1, R2 and R4 cooling systems were the most efficient in preserving sperm viability, membrane integrity and intracellular H2O2. Samples frozen in the C1 system exhibited better post-thaw and post-TRT kinetics than C2 samples. In conclusion, slower cooling curves in conjunction with a constant freezing rate obtained with the programmable unit were more efficient for freezing bull semen in the field.

Effect of seminal plasma removal before cryopreservation of bovine semen obtained by electroejaculation on semen quality and in vitro fertility.

Cryopreservation of bull semen is a common biotechnology procedure in cattle breeding. However, when the ejaculate is obtained by electroejaculation, wide variation is observed in the sperm/seminal plasma (SP) ratio that can affect the freezability of semen in this species. The removal of SP may improve the quality of frozen bull semen. The objective of this study was to evaluate the effect of SP removal from the ejaculate on the cryopreservation of semen from 38 Nellore bulls collected by electroejaculation. After collection, the ejaculate was divided into three aliquots: (1) control (N) diluted to a concentration of 60 × 106 spermatozoa/mL and frozen with SP; (2) centrifugation (C) at ×600g for 10 minutes and the pellet resuspended and frozen at the same concentration as N; and (3) filtration (F) through SpermFilter and sperm recovered and frozen at the same concentration as N. After thawing, sperm kinetics, plasma and acrosome membrane integrity, mitochondrial membrane potential, oxidative stress, and in vitro fertility were evaluated. Statistical analysis was performed using the SAS 9.2 package, and differences were considered significant when P < 0.05. Higher average path velocity and straight-line velocity were observed in the groups submitted to SP removal compared to the control group (P < 0.01). In contrast, filtered samples exhibited higher beat cross frequency, straightness, and linearity compared to the other groups. Plasma membrane integrity was reduced when SP was removed, but lower oxidative stress was observed in groups C and F (34.91 ± 2.95% and 31.63 ± 2.95%, respectively) compared to group N (57.39 ± 2.95%). However, the percentage of hatched blastocysts was similar in the N and F groups (21.22 ± 1.05% and 24.00 ± 1.05%, respectively) and higher compared to group C (18.83 ± 1.05%). In conclusion, removal of SP by centrifugation for bull semen freezing reduced the rate of in vitro-produced embryos, whereas filtration of prefrozen semen was found to be an efficient alternative in terms of semen freezability and in vitro production of bovine embryos.

Assessment of different in vitro sperm challenges and in vivo fertility of bovine semen batches / Avaliação de diferentes desafios espermáticos in vitro e da fertilidade in vivo de partidas de sêmen bovino

The aim of this work was to submit sperm cells to different laboratory challenges and to compare in vitro results with in vivo semen fertility. Four different batches from the same Brangus bull were used in a timed-AI program of 332 Brangus cows. Each batch (B) was submitted to the following procedure: semen sample was thawed at 36°C for 30 seconds (control). Sperm motility parameters, plasma membrane integrity, sperm morphology, and concentration were assessed. Then, an aliquot of thawed sample was incubated in a water bath at 45°C for 40 min (thermal challenge group; TCG) and another aliquot was centrifuged at 500 xg (Percoll gradient 45%/90%) for 15 min (centrifugation challenge group; CCG). Centrifuged semen was also submitted to another thermal challenge, being incubated (water bath) at 45°C for 40 min (centrifugation + thermal challenge group; CTCG). At the end of each challenge (CCG, TCG, and CTCG), the same laboratory tests used for control group were repeated. The following conception rates (CR) were observed for each batch: B1 = 48.9% (44/90); B2 = 44.2% (23/52); B3 = 55.5% (40/72); B4 = 43.2% (51/118); (p < 0.10). In the lab, B3 presented higher (p ≤ 0.05) progressive motility (PM) than B4 after thawing (control group) and after all sperm challenges (TCG, CCG, and CTCG). However, despite B3 and B4 having demonstrated a similar percentage of plasma membrane integrity (PMI) to the control group (B3 = 66.7 ± 1.3 and B4 = 65.2 ± 3.3), B3 demonstrated higher (P ≤ 0.05) percentage of PMI (37.2 ± 2.5) than B4 (26.7 ± 3.3) after passing through the most stressing in vitro challenge (CTCG). The semen batch presenting the highest resistance to in vitro challenges was the one that presented a trend for higher in vivo fertility, suggesting that submitting semen samples to laboratory challenges may be an interesting alternative for selecting batches with greater field fertility.(AU)

O objetivo deste estudo foi estressar células espermáticas em diferentes desafios laboratoriais e comparar os resultados in vitro com a fertilidade in vivo do sêmen. Quatro partidas de um mesmo touro Brangus foram utilizadas em um programa de IATF de 332 vacas Brangus. Cada partida foi submetida ao seguinte procedimento: a amostra de sêmen foi descongelada a 36°C por 30 segundos (grupo controle). Foram avaliados parâmetros de motilidade espermática (CASA), integridade da membrana plasmática (PMI), morfologia e concentração espermática. Em seguida, uma alíquota da amostra descongelada foi incubada em banho-maria a 45°C durante 40 minutos (grupo de desafio térmico, TCG) e outra alíquota foi centrifugada a 500 xg (gradiente de Percoll 45%/90%) durante 15 min (grupo desafio de centrifugação, CCG). Uma aliquota do sêmen centrifugado foi ainda submetida ao desafio térmico, sendo incubado a 45°C durante 40 min (grupo de desafio térmico + centrifugação, CTCG). No final de cada desafio (CCG, TCG e CTCG), os mesmos testes laboratoriais utilizados para o grupo de controle foram realizados. A seguinte taxa de concepção (CR) foi observada para cada partida (B): B1 = 48,9% (44/90), B2 = 44,2% (23/52), B3 = 55,5% (40/72) e B4 = 43,2% (51/118); (P < 0,10). No laboratório, B3 apresentou maior (P ≤ 0,05) motilidade progressiva (PM) do que B4 logo após o descongelamento (grupo controle) e após todos os desafios laboratoriais (TCG, CCG e CTCG). Porém, apesar de B3 e B4 demonstrarem similar porcentagem de PMI no grupo controle (B3 = 66,7 ± 1,3 e B4 = 65,2 ± 3,3), B3 apresentou maior (P ≤ 0,05) PMI (37,2 ± 2,5%) do que B4 (26,7 ± 3,3%) após passar pelo maior desafio laboratorial (CTCG). A partida seminal que in vitro apresentou maior resistência aos desafios laboratoriais foi a mesma que apresentou tendência para maior fertilidade in vivo. Assim, sugere-se que submeter amostras seminais a desafios laboratoriais pode ser uma alternativa interessante para selecionar partidas com maior fertilidade a campo.(AU)