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The study aimed to assess the effect of long-acting bST treatment, in a dose that only increases IGF-I plasma concentrations, on ovarian and fertility markers of estrous synchronized ewes that were fed to keep their bodyweight. Three experiments were designed to evaluate this effect: in Experiment 1, 18 ewes were distributed in groups (bST 0, 30, 50 mg) to measure plasma IGF-I and insulin for 15 days; in Experiment 2, 92 ewes (5 replicates) in two groups (0 and 30 mg bST) were synchronized using a 6-day progesterone protocol during the breeding season to assess the effect of bST on follicular and luteal performances, estrous and ovulation, and fertility after mating. In Experiment 3, 50 ewes (3 replicates) were used to repeat the study before but during anestrus. Results indicate that 50 mg bST increased IGF-I and insulin plasma concentrations, but 30 mg bST only increased IGF-I concentrations; and that only during the breeding season did 30 mg bST increase the number of lambs born and the reproductive success of ovulatory-sized follicles compared to controls. This occurred without it affecting any other reproductive marker. In conclusion, 30 mg bST treatment may improve oocyte competence for fertility during the breeding season.

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The study tested the hypothesis that a single administration of hCG supports the LH-dependent phase of terminal follicular development in synchronized sheep during anestrus, using eCG as a functional reference. Using a clinical approach, four experiments were designed to achieve the following: (1) Identify the inhibitory influence of anestrus on reproduction efficiency; (2) Assess the potential of hCG to keep functional blood concentrations after a single dose; (3) Characterize the effect of different doses of hCG on reproductive functional markers; (4) To compare the ability of hCG to that of eCG to support follicular development and fertility based on the same markers. The results showed that anestrus seems to affect follicular and luteal function under LH dependency as FSH-dependent markers are not compromised; hCG maintains higher blood concentrations than controls for at least 48 h; hCG improves follicular development and ovulatory rates compared to controls and at standards comparable to a breeding season; and ewes treated with hCG exhibit similar performance to those treated with eCG. Our results conclude that hCG can be used to support follicular function during anestrus in sheep, aiming to perfect its regulation in assisted reproduction.

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l-carnitine (LC) transports fatty acids to the mitochondria for energy production, reducing lipid availability for peroxidation through ß-oxidation. This research examines the effect of LC supplementation to two skimmed milk-based extenders on the cryosurvival of chilled (5°C) and frozen-thawed Peruvian Paso horse spermatozoa .An initial experiment determined the optimal LC concentration (0, 1, 5, 10, 25, and 50 mM) when added to INRA-96® and UHT (skimmed milk + 6% egg yolk) extenders, using nine ejaculates from three stallions chilled for up to 96 h. Subsequently, the effect of 25 mM LC supplementation (the optimal concentration) on chilling (INRA-96) and freezing (INRA-Freeze®) extenders was evaluated using eight pooled samples from sixteen ejaculates (2 ejaculates/pool) from four stallions. Results indicated that all LC concentrations produced significantly higher values (P<0.05) for kinematic variables (total [TM] and progressive motilities, curvilinear [VCL] and straight-line [VSL] velocity, and beat-cross frequency [BCF]), and the integrity of plasma/acrosome membranes (IPIA) compared to non-supplemented chilled sperm samples for up to 96 h with both extenders. Moreover, the use of 25 mM LC was more efficient (P<0.05) in preserving the post-chilled values of velocity, BCF, and IPIA for the long term than lower LC concentrations (1-10 mM). Post-thaw values of total motility, the amplitude of lateral head displacement (ALH), and IPIA were significantly improved (P<0.05) when INRA-Freeze extender was supplemented with 25 mM LC. In conclusion, supplementation of l-carnitine to skimmed milk-based extenders enhanced kinematic variables and protected the membrane integrity in chilled and frozen-thawed Peruvian Paso horse spermatozoa.

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Equus members exhibit very divergent karyotype, genetic plasticity, and significant differences in their reproductive physiology. Despite the fact that somatic cell nuclear transfer and intracytoplasmic sperm injection (ICSI) has gained relevance in the last few years in horses, few reports have been published exploring ovum pick up (OPU) and in vitro maturation (IVM) of cumulus-oocyte complexes (COCs) in donkeys. Yet, some donkey species and breeds are considered endangered, and these assisted-reproductive technologies could help to preserve the genetic of valuable individuals. In this study, we tested the hypothesis that supplementation with jenny preovulatory follicular fluid (PFF) during IVM could improve oocyte developmental competence in the donkey. For this, in vitro nuclear maturation rates, cumulus cell expansion, and embryo development after ICSI of donkey COCs matured in culture media supplemented with fetal bovine serum (FBS) or donkey PFF, with a known metabolomic profile, were assessed. Time-lapse imagining was performed after ICSI of horse and donkey oocytes. Eight OPU sessions were done in five jennies with an average recovery rate of 69.2% (n = 45 COCs). Although lower cumulus cells expansion was observed in oocytes of PFF group (P = 0.0010), no significant differences were described in nuclear maturation rates and preimplantation embryo development between groups. Donkey ICSI embryos showed similar morphokinetics to horse ICSI embryos. Our study shows that supplementing IVM media with FBS or donkey PFF supports nuclear maturation and early preimplantation embryo development after ICSI in donkeys. To our knowledge, the present study is the first report of ICSI, time-lapse imaging and in vitro blastocyst production in donkey.

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This research examined the antioxidant and cryoprotective effects of melatonin (ME) and caffeine (CAF) supplementation in freezing medium on the cryosurvival of Peruvian Paso horse sperm using a two-step accelerating cooling rate. Twenty ejaculates from four adult and fertile stallions were recovered, initially diluted with INRA-96®, and finally frozen with INRA-Freeze® with either no supplementation (as control), 1 µM ME, or 2 mM CAF using a two-ramp freezing system content inside a cryogenic-box and liquid nitrogen vapors. The sperm kinematic parameters and integrity of the plasma and acrosomal membranes of fresh semen and cryopreserved samples were evaluated using the CASA system (SCA-Evolution® 2018) and PI/fluorescein isothiocyanate-conjugated peanut (Arachis hypogaea) agglutinin double fluorescent test, respectively. The oxidative stress of post-thaw sperm samples was also assessed using the CellRox Deep Red fluorescence test. The results showed that curvilinear velocity and average-path velocity were greater (p < 0.05) after freezing with CAF than the control group. In addition, there were significance differences (p < 0.01) between stallions (1-4) in post-thaw kinematic parameters regardless of ME or CAF addition. Both ME and CAF improved (p < 0.05) the proportion of sperm with intact plasma membranes and intact acrosomes. Nevertheless, neither CAF nor ME improved the oxidative stress after the cryopreservation process.

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The effect of inbreeding depression on sperm motility is well documented, but its influence on sperm morphometry has been scarcely examined to date. Here, we combined the use of computer-assisted sperm morphometry analysis (CASMA) with a SNP-based genomic approach to determine and characterize the effect of inbreeding on the sperm shape of a highly inbred cattle population. We determined seven morphometric parameters on frozen-thawed sperm samples of 57 Retinta bulls: length (L, µm), width (W, µm), area (A, µm2 ), perimeter (P, µm), ellipticity (ELI; L/W), elongation (L-W)/(L + W) and perimeter-to-area shape factor (p2a; P2 /4 × π × A). The comparison of highly inbred (HI) and lowly inbreed (LI) individuals based on runs of homozygosity (ROH) inbreeding values (F ROH ) showed no differences between groups. An additional two-step unsupervised sperm subpopulation analysis based on morphometric parameters showed significant differences in the abundance of different sperm subpopulations between groups (p < 0.05). This analysis revealed that HI bulls harbored a higher percentage of narrow-head sperm as opposed to the higher percentage of large- and round-headed sperm detected in LI. A further genomic characterization revealed 23 regions differentially affected by inbreeding in both groups, detecting six genes (SPAG6, ARMC3, PARK7, VAMP3, DYNLRB2, and PHF7) previously related to different spermatogenesis-associated processes.

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The aim of this study was to compare the post-thaw distribution of motile sperm subpopulations, following simple or colloid centrifugation. A new analysis was used to evaluate the available number of sperm from each subpopulation after each centrifugation protocol. Frozen/thawed semen samples were divided into the following after-thawing treatments: uncentrifuged control (UDC), sperm washing (SW) and two colloid centrifugation procedures (Equipure, SLC-E, and Androcoll, SLC-A). Percentage of total and progressive motility (TM and PM), as well as sperm motility kinematics, distribution of motile sperm subpopulations, and recovery rates, were statistically compared among treatments. The SLC treatments showed higher (P < 0.001) TM and PM than UDC and SW. Following each SLC procedure, different percentages of the subpopulation with the most vigorous and progressive sperm (sP4) were obtained. SLC-A recovered a larger number of sperm belonging to sP4 than SLC-E, but not significantly higher than SW. From a practical point of view, sperm washing, the standard centrifugation procedure for equine semen processing, recovered the same amount of fast and progressive sperm as colloid centrifugation, apparently the best treatment according to traditional analysis. In conclusion, samples processed by SLC have higher motility percentages than SW and UDC but, after combining the available number of sperm, SLC and SW techniques are equally efficient in recovering sperm from the most vigorous, fast and progressive motile subpopulation (sP4).

RESUMO

The aim of this study was to compare the postthaw distribution of motile sperm subpopulations, following simple or colloid centrifugation. A new analysis was used to evaluate the available number of sperm from each subpopulation after each centrifugation protocol. Frozen/thawed semen samples were divided into the following after-thawing treatments: uncentrifuged control (UDC), sperm washing (SW) and two colloid centrifugation procedures (Equipure, SLCE, and Androcoll, SLC-A). Percentage of total and progressive motility (TM and PM), as well as sperm motility kinematics, distribution of motile sperm subpopulations, and recovery rates, were statistically compared among treatments. The SLC treatments showed higher (P < 0.001) TM and PM than UDC and SW. Following each SLC procedure, different percentages of the subpopulation with the most vigorous and progressive sperm (sP4) were obtained. SLC-A recovered a larger number of sperm belonging to sP4 than SLC-E, but not significantly higher than SW. From a practical point of view, sperm washing, the standard centrifugation procedure for equine semen processing, recovered the same amount of fast and progressive sperm as colloid centrifugation, apparently the best treatment according to traditional analysis. In conclusion, samples processed by SLC have higher motility percentages than SW and UDC but, after combining the available number of sperm, SLC and SW techniques are equally efficient in recovering sperm from the most vigorous, fast and progressive motile subpopulation (sP4).(AU)

Assuntos

RESUMO

The aim of this study was to compare the postthaw distribution of motile sperm subpopulations, following simple or colloid centrifugation. A new analysis was used to evaluate the available number of sperm from each subpopulation after each centrifugation protocol. Frozen/thawed semen samples were divided into the following after-thawing treatments: uncentrifuged control (UDC), sperm washing (SW) and two colloid centrifugation procedures (Equipure, SLCE, and Androcoll, SLC-A). Percentage of total and progressive motility (TM and PM), as well as sperm motility kinematics, distribution of motile sperm subpopulations, and recovery rates, were statistically compared among treatments. The SLC treatments showed higher (P < 0.001) TM and PM than UDC and SW. Following each SLC procedure, different percentages of the subpopulation with the most vigorous and progressive sperm (sP4) were obtained. SLC-A recovered a larger number of sperm belonging to sP4 than SLC-E, but not significantly higher than SW. From a practical point of view, sperm washing, the standard centrifugation procedure for equine semen processing, recovered the same amount of fast and progressive sperm as colloid centrifugation, apparently the best treatment according to traditional analysis. In conclusion, samples processed by SLC have higher motility percentages than SW and UDC but, after combining the available number of sperm, SLC and SW techniques are equally efficient in recovering sperm from the most vigorous, fast and progressive motile subpopulation (sP4).

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Treintra y siete vacas con historia de repetición de celos fueron sometidas a exploraciones ecográficas de su aparato genital y a determinación de progesterona plasmática desde el día 0 del ciclo (día en que se realizó la espermatización) hasta el día 32, a intervalos de cuatro días. Se observó que un 16.2 por ciento de estos animales presentaba estructuras quísticas ováricas, diferenciándose un 70 por ciento de naturaleza folicular, con aspecto ecográfico anecogénico, paredes delgadas hiperecogénicas y que aparecían solos (quiste folicular simple) o agrupados (quistes foliculares múltiples); y un 30 por ciento de naturaleza luteínica, con morfología redondeada, cuya apariencia ecográfica era anecogénica y a veces mostraba un entramado hipoecogénico, con paredes a menudo engrosadas. Así mismo, el curso clínico consistía en presentación de celos cortos y frecuentes en un 66.6 por ciento de los animales, mientras que el 33.3 por ciento mostró anestro o pérdida del comportamiento estral. Se determinó la concentración de progesterona a lo largo del periodo de estudio, diferenciándose vacas con niveles basales de progesterona (menor de 0.8 ng/ml), otras con niveles suprabasales (entre 0.8 y 1.8 ng/ml), y animales que alternaron valores altos y bajos dependiendo de la naturaleza del quiste y del momento en que se había formado.

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