Effect of seminal plasma cholesterol and triacylglycerides concentrations and sperm morphology on semen freezability in domestic cats (Felis silvestris catus).

There is scarce information about the effect of sperm morphology and seminal plasma composition on cat semen freezability. Thus, this study aims to assess the effect of cat sperm morphology and seminal plasma cholesterol (CHOL) and triacylglyceride (TAG) concentrations on sperm post-thaw survival. Ejaculates (n = 49) were evaluated, and seminal plasma was separated and frozen until CHOL and TAG concentrations were measured. The sperm pellet was diluted in a tris-based egg yolk extender, frozen (n = 38), or processed for sperm ultrastructure study (n = 11). Abnormalities recorded were abnormal head shape and size, detached heads, knobbed or ruffled acrosomes, eccentric mid-piece insertion, proximal and distal cytoplasmic droplets, folded and coiled tails, and Dag defect. Ultramicroscopic evaluation detected several sperm abnormalities in fresh semen and some sperm damage in frozen semen. Seminal plasma lipids components were positively correlated with post-thaw motility and acrosome integrity. Higher freezability indices for motility and acrosome integrity were observed in frozen-thawed semen with high seminal plasma CHOL and TAG concentrations. No freezability differences were observed between teratozoospermic and normozoospermic ejaculates. Our results showed that even when seminal plasma was removed before cryopreservation, sperm survival after thawing was significantly higher in samples with high seminal plasma CHOL and TAG concentrations, indicating a rapid adherence to these compounds to the sperm plasma membrane, protecting sperm cells from temperature changes. Nevertheless, there were no differences in sperm freezability by sperm morphology.

Pregnancy and birth rate outcomes in alpacas (Vicugna pacos) inseminated with frozen semen using two commercial extenders.

The aim of this study was to evaluate alpaca pregnancy outcomes and birth rates of females inseminated with frozen semen using two commercial extenders. A total of 18 ejaculates from 8 adult alpaca males were obtained with artificial vagina, and macroscopic and microscopic semen characteristics were assessed. Afterwards, samples were divided into two aliquots, diluted with Biladyl® B or AndroMed®, and cooled for 2 h at 5°C. At that moment, sperm motility was evaluated, and samples were frozen through a gradual descent of temperature using a liquid nitrogen tank. To analyse frozen sperm quality, samples were thawed at 38°C for 30 s. Even though a significant decrease in sperm motility and viability was detected when thawed (p < .05), no superiority was found between the two commercial extenders (Biladyl® B vs. AndroMed®). A total of 36 alpaca females were artificially inseminated (AI) between 30 and 34 h post-injection of a GnRH analogue, administered when a growing dominant follicle was detected through transrectal palpation and ultrasonography. Obtained pregnancy rates were similar between Biladyl® B (33.3%, 6/18) and AndroMed® (22.2%, 4/18). No significant differences were detected in birth rates between the two tested extenders, obtaining 4 and 3 births for Biladyl® and AndroMed®, respectively. In conclusion, alpaca pregnancies and alive offspring can be obtained through AI with frozen semen at similar efficiency rates using commercial diluents, Biladyl® B or AndroMed®.

Reprodução ex situ de felinos silvestres e seus desafios / Challenges of ex situ reproduction in wild cats

A reprodução ex situ de felinos silvestres assegura a sobrevivência das espécies ameaçadas por meio do estabelecimento e manutenção de populações viáveis em cativeiro. O conhecimento da biologia reprodutiva básica é essencial para o desenvolvimento de planos de manejo reprodutivo (PMRs) eficientes, seja pela reprodução natural ou pela aplicação de técnicas de reprodução assistida (TRAs). Os PMRs visam garantir a representatividade das espécies quanto à variabilidade genética e demográfica baseada nos studbooks. Entretanto, o pareamento de animais selecionados pelos PMRs deve levar em conta, além de fatores genéticos e demográficos, fatores comportamentais e o fenótipo dos animais, uma vez que pode haver consequências negativas caso descendentes de gerações futuras sejam reintroduzidos na natureza. As TRAs estão cada vez mais sendo desenvolvidas para auxiliar na manutenção de populações geneticamente viáveis ex situ que possam contribuir geneticamente com populações in situ. A criopreservação de sêmen e a inseminação artificial (IA) são as TRAs utilizadas atualmente pelos PMR nacionais e internacionais, no entanto, são muitos os desafios para que as populações cativas se reproduzam de maneira adequada visando a manutenção de uma população viável que possa contribuir com populações de vida livre no futuro.(AU)

Reproductive management plans are essential to ensure that imperiled populations maintain adequate genetic and demographic variability and remain representative of the species as a whole. Basic reproductive biology knowledge is essential for the development of efficient reproductive management plans (PMPs), either through natural breeding or through assisted reproductive techniques (ARTs). The PMPs aim to ensure the representativeness of the species in terms of genetic and demographic variability based on studbooks. However, the specific animal pairings should be maintaining adequate genetic, behavioral and the phenotype of the animals, ensuring proper reintroduction of animals into the wild. ARTs have been explored as a means to enhance the conservation of endangered species, focused on maintaining genetic diversity through enhanced animal propagation. Semen cryopreservation and artificial insemination (AI) are used by national and international PMRs, however, there are many challenges for captive populations reproduction in order to maintain a viable population that can contribute for freeliving populations in the future.(AU)

Heterologous in vitro fertilization and embryo production for assessment of jaguar (Panthera onca Linnaeus, 1758) frozen-thawed semen in different extenders.

Heterologous in vitro fertilization (IVF) is an important tool for assessing fertility of endangered mammals such as the jaguar, considering difficult access to females for artificial insemination and to obtain homologous oocytes. We aimed to evaluate the fertility of jaguar sperm cryopreserved with different extenders, using domestic cat oocytes to assess the development of hybrid embryos. Semen from four captive jaguars was obtained by electroejaculation. Samples were cryopreserved in powdered coconut water (ACP-117c) or Tris extender containing 20% egg yolk and 6% glycerol. Thawed spermatozoa were resuspended (2.0 × 106 spermatozoa/mL) in IVF medium and co-incubated with cat oocytes matured in vitro for 18 h. Presumptive zygotes were cultured for 7 days. After 48 h, cleavage rate was evaluated, and non-cleaved structures were stained for IVF evaluation. On days 5 and 7, the rate of morula and blastocyst formation was assessed. Data were analyzed using the Fisher exact test (p < 0.05). No difference was observed between ACP-117c and Tris extenders, respectively, for oocytes with 2nd polar body (2/51, 3.9 ± 2.9% vs. 2/56, 3.6 ± 3.1%), pronuclear structures (5/51, 9.8 ± 4.7% vs. 8/56, 14.3 ± 8.0%), and total IVF rates (7/36, 19.4 ± 5.0% vs. 10/37, 27.0 ± 13.8%). All the samples fertilized the oocytes, with 22.9 ± 3.2% (16/70) and 16.7 ± 3.6% (12/72) cleavage of mature oocytes for ACP-117c and Tris extenders, respectively. Morula rates of 4.3 ± 2.3% (3/70) and 5.6 ± 2.2% (4/72) were observed for ACP-117c and Tris, respectively. Only the Tris extender demonstrated blastocyst production (2/12, 16.7 ± 1.5% blastocyst/cleavage). We demonstrated that jaguar ejaculates cryopreserved using ACP-117c and Tris were suitable for IVF techniques, with blastocyst production by ejaculates cryopreserved in Tris. This is a first report of embryos produced in vitro using jaguar sperm and domestic cat oocytes through IVF.

Relationship between sperm ubiquitination and equine semen freezability.

This study aimed to assess the semen ubiquitin levels of stallions with good (GF) and poor semen freezability (PF) and to evaluate the relationship between sperm ubiquitination and sperm morphological defects. Five ejaculates from eight adult stallions (n = 40) were collected and cryopreserved. Then, the ubiquitin level in equine sperm cells was assessed by immunohistochemistry with epifluorescence microscopy, and sperm morphology was assessed by differential interference contrast microscopy. Sperm cells were classified according to the intensity (classification 1: from I to IV; I = very low ubiquitin intensity and IV = very high ubiquitin intensity) and location of ubiquitin staining (classification 2). Statistical analyses were performed using SAS software (version 9.4), and p ≤ .05 was considered significant. We observed that PF stallions showed higher percentages (p < .05) of sperm cells with high ubiquitination (11.82% of ubiquitin intensity grade I, 39.13% of ubiquitin intensity grade II, 27.25% of ubiquitin intensity grade III, and 20.67% of grade IV), while GF stallions showed higher percentages (p < .05) of sperm cells with lower staining intensity (28.52% grade I, 59.83% grade II, 7.92% grade III, and 7.02% grade IV). Furthermore, for PF stallions, 23 significant correlations were detected (p < .05) between sperm abnormalities and ubiquitin intensity in different sperm regions. Increased ubiquitination of the sperm head, midpiece, and tail was positively correlated with their respective morphological defects. We concluded that high sperm ubiquitin levels are observed in ejaculates from stallions with poor semen quality (poor freezability), and ubiquitin marking in specific cellular locations can identify sperm morphological defects.

Heterologous in vitro fertilization and embryo production for assessment of jaguar (Panthera onca Linnaeus, 1758) frozen-thawed semen in different extenders

Heterologous in vitro fertilization (IVF) is an important tool for assessing fertility of endangered mammals such as the jaguar, considering difficult access to females for artificial insemination and to obtain homologous oocytes. We aimed to evaluate the fertility of jaguar sperm cryopreserved with different extenders, using domestic cat oocytes to assess the development of hybrid embryos. Semen from four captive jaguars was obtained by electroejaculation. Samples were cryopreserved in powdered coconut water (ACP-117c) or Tris extender containing 20% egg yolk and 6% glycerol. Thawed spermatozoa were resuspended (2.0 × 106 spermatozoa/mL) in IVF medium and co-incubated with cat oocytes matured in vitro for 18 h. Presumptive zygotes were cultured for 7 days. After 48 h, cleavage rate was evaluated, and non-cleaved structures were stained for IVF evaluation. On days 5 and 7, the rate of morula and blastocyst formation was assessed. Data were analyzed using the Fisher exact test (p < 0.05). No difference was observed between ACP-117c and Tris extenders, respectively, for oocytes with 2nd polar body (2/51, 3.9 ± 2.9% vs. 2/56, 3.6 ± 3.1%), pronuclear structures (5/51, 9.8 ± 4.7% vs. 8/56, 14.3 ± 8.0%), and total IVF rates (7/36, 19.4 ± 5.0% vs. 10/37, 27.0 ± 13.8%). All the samples fertilized the oocytes, with 22.9 ± 3.2% (16/70) and 16.7 ± 3.6% (12/72) cleavage of mature oocytes for ACP-117c and Tris extenders, respectively. Morula rates of 4.3 ± 2.3% (3/70) and 5.6 ± 2.2% (4/72) were observed for ACP-117c and Tris, respectively. Only the Tris extender demonstrated blastocyst production (2/12, 16.7 ± 1.5% blastocyst/cleavage). We demonstrated that jaguar ejaculates cryopreserved using ACP-117c and Tris were suitable for IVF techniques, with blastocyst production by ejaculates cryopreserved in Tris. This is a first report of embryos produced in vitro using jaguar sperm and domestic cat oocytes through IVF.(AU)

Avanços na andrologia de grandes felinos neotropicais / Advances in neotropical big cats andrology

O desenvolvimento de tecnologias reprodutivas e conhecimentos sobre a andrologia de grandes felinos caminham a pequenos passos, com avanços na última década. Estudos sobre o comportamento de onças-pintadas (Panthera onca) revelam as sequências de comportamentos sócio-sexuais e levantam a possibilidade de a ovulação poder ocorrer por estímulos sensoriais, e não somente pela estimulação mecânica durante a cópula. Um grande avanço na andrologia dos felinos foi o desenvolvimento da colheita farmacológica com α2-agonistas, que já se mostrou eficiente também nos grandes felinos neotropicais. Este foi um verdadeiro divisor de águas para a criopreservação de sêmen, especialmente em animais de vida livre. Pouco se avançou, no entanto, no meio de criopreservação, ainda hoje os meios indicados são à base de TRIS-gema-glicerol, porém a gema não é estável suficiente para uso em animais de vida livre, sendo necessária avaliação de substitutivos como as lipoproteínas de baixa densidade e lecitina de soja. O aprimoramento de reprodução assistida nos felinos neotropicais é pungente, em especial a onça-pintada visto que em alguns biomas a espécie está criticamente em perigo.

The development of reproductive technologies and knowledge about the andrology of big cats are taking small steps, with advances in the last decade. Studies on the sexual behavior of jaguars (Panthera onca) reveal the sequences of sexual behaviors and raise the possibility that ovulation may occur through sensory stimuli and not only through mechanical stimulation during copulation. A significant advance in feline andrology was the development of pharmacological semen collection with α2-agonists, which has proved efficient in neotropical big cats. It was disruptive for semen cryopreservation, especially in free-living animals. Little progress has been made; however, in the cryopreservation environment, even today, the indicated means are based on TRIS-yolk-glycerol. However, the yolk is not stable enough for use in free-living animals, requiring the evaluation of substitutes such as low-density lipoproteins and soy lecithin. The improvement of assisted reproduction in neotropical felines is poignant, especially the jaguar, since in some biomes, the species is critically endangered.

Semen Cryopreservation and Banking for the Conservation of Neotropical Carnivores.

Neotropical carnivores include a large number of threatened and endangered species. It is critical to develop conservation efforts to ensure the sustainability of populations in situ and ex situ. The highest priorities are to protect natural habitats and better understand the biology of rare species. Conservation efforts also are directed toward the implementation of breeding programs and the development of reproductive biotechnologies in which the cryopreservation of male gametes plays a major role. It also is fundamental to create semen banks that contribute to maintaining genetic diversity in small and endangered populations. The present article aims at reviewing the state of the art in cryopreservation of semen from neotropical carnivores and discuss the development of systematic banking for the conservation of these understudied species.

Efeito de meios diluentes e sistemas de transporte no sêmen congelado analisado pelo método computacional CASA em touros Nelore / Effect of extenders and transportation systems in frozen semen analyzed by computational method HOME in Nelore bulls

Objetivou-se avaliar meios diluentes e sistemas de transporte na qualidade do sêmen congelado pelo CASA em touros Nelore. Foram realizadas cinco coletas de sêmen de 6 touros, diluídas com os meios TRIS e BotuBOV®, refrigeradas em dois tipos de sistemas de transporte BotuBOX® e BotuFLEX®; congeladas e analisadas pelo CASA. A MT foi maior (p<0,05) na associação do meio BotuBOV® + BotuFLEX®(47,3%) quando comparado ao TRIS + BotuBOX® (9%). A MP foi maior (p<0,05) BotuBOV® + BotuFLEX® (37%) quando comparada a TRIS + BotuBOX® (5,9%). O mesmo para VSL, BCF e RAP, os quais foram maiores (p<0,05) em BotuBOV® + BotuFLEX® (65,1μm/s; 30Hz; 44,5%, respectivamente) quando comparados ao sêmen na associação TRIS + BotuBOX® (47,6μm/s; 21,5Hz; 6,9%, respectivamente). Conclui-se que o BotuBOV® em associação ao sistema de transporte refrigerado de sêmen BotuFLEX®, foi considerada pelo CASA, a melhor associação quando se refere a cinética espermática para a criopreservação de sêmen bovino.(AU)

The objective was to evaluate diluents and transport systems on semen quality frozen by CASA in Nellore bulls. Five semen collections of six bulls, diluted with the TRIS and BotuBOV®, were transported out on two types of BotuBOX® and BotuFLEX® systems; frozen and analyzed by CASA. TM was higher (p <0.05) in the association of BotuBOV® + BotuFLEX® (47.3%) when compared to TRIS + BotuBOX® (9%). The PM was higher (p <0.05) BotuBOV® + BotuFLEX® (37%) when compared to TRIS + BotuBOX® (5.9%). The same for VSL, BCF and RAP, which were higher (p<0.05) in BotuBOV® + BotuFLEX® (65.1μm/s, 30Hz, 44.5%, respectively) when compared to semen in the TRIS + BotuBOX® (47.6μm/s, 21.5Hz, 6.9%, respectively). It is concluded that BotuBOV® in association with the refrigerated system of semen BotuFLEX® was considered by CASA to be the best association when referring to sperm kinetics for cryopreservation of bovine semen.(AU)

Efeito de meios diluentes e sistemas de transporte no sêmen congelado analisado pelo método computacional CASA em touros Nelore / Effect of extenders and transportation systems in frozen semen analyzed by computational method HOME in Nelore bulls

Objetivou-se avaliar meios diluentes e sistemas de transporte na qualidade do sêmen congelado pelo CASA em touros Nelore. Foram realizadas cinco coletas de sêmen de 6 touros, diluídas com os meios TRIS e BotuBOV®, refrigeradas em dois tipos de sistemas de transporte BotuBOX® e BotuFLEX®; congeladas e analisadas pelo CASA. A MT foi maior (p<0,05) na associação do meio BotuBOV® + BotuFLEX®(47,3%) quando comparado ao TRIS + BotuBOX® (9%). A MP foi maior (p<0,05) BotuBOV® + BotuFLEX® (37%) quando comparada a TRIS + BotuBOX® (5,9%). O mesmo para VSL, BCF e RAP, os quais foram maiores (p<0,05) em BotuBOV® + BotuFLEX® (65,1μm/s; 30Hz; 44,5%, respectivamente) quando comparados ao sêmen na associação TRIS + BotuBOX® (47,6μm/s; 21,5Hz; 6,9%, respectivamente). Conclui-se que o BotuBOV® em associação ao sistema de transporte refrigerado de sêmen BotuFLEX®, foi considerada pelo CASA, a melhor associação quando se refere a cinética espermática para a criopreservação de sêmen bovino.

The objective was to evaluate diluents and transport systems on semen quality frozen by CASA in Nellore bulls. Five semen collections of six bulls, diluted with the TRIS and BotuBOV®, were transported out on two types of BotuBOX® and BotuFLEX® systems; frozen and analyzed by CASA. TM was higher (p <0.05) in the association of BotuBOV® + BotuFLEX® (47.3%) when compared to TRIS + BotuBOX® (9%). The PM was higher (p <0.05) BotuBOV® + BotuFLEX® (37%) when compared to TRIS + BotuBOX® (5.9%). The same for VSL, BCF and RAP, which were higher (p<0.05) in BotuBOV® + BotuFLEX® (65.1μm/s, 30Hz, 44.5%, respectively) when compared to semen in the TRIS + BotuBOX® (47.6μm/s, 21.5Hz, 6.9%, respectively). It is concluded that BotuBOV® in association with the refrigerated system of semen BotuFLEX® was considered by CASA to be the best association when referring to sperm kinetics for cryopreservation of bovine semen.

Capacidad antioxidante y calidad post-descongelación del semen equino criopreservado con quercetina y ergotioneina / Antioxidant capacity and post-thaw quality of stallion semen cryopreserved with quercetin and ergothioneine

Se evaluó la capacidad antioxidante y la calidad post-descongelación del semen equino criopreservado con quercetina y ergotioneina. Nueve eyaculados provenientes de tres caballos criollos colombianos se criopreservaron bajo tres tratamientos: ergotioneina (100 pM), quercetina (100 pM) y control (sin antioxidante). Posteriormente a la descongelación se evaluaron los siguientes parámetros: la capacidad antioxidante total (CAT) del semen mediante el ensayo del ácido 2,2'-azino-bis-[3-etilbenzotiazolina]-6-sulfónico (ABTS•+); la movilid ad total (MT); la movilidad progresiva (MP); la hiperactividad (HA) y las velocidades curvilínea (VCL), lineal (VSL) y media (VAP) mediante el sistema computarizado SCA ; además, la integridad estructural de la membrana y la integridad acrosómica por microscopia de fluorescencia mediante las sondas SYBR/IP y FITC/ PNA, respectivamente; la morfología mediante la tinción eosina-nigrosina y la integridad funcional de membrana a través de la prueba hipoosmótica (HOS). Se realizó el ajuste de modelos lineales generalizados (GLM) y la comparación de medias por Tukey. La CAT (pmol trolox/ml) del semen descongelado fue superior para la ergotioneina (4,0 ± 0,3) y la quercetina (3,9 ± 0,4), respecto del control (2,6 ± 1,5). Para la MT se encontró una media superior para la ergotioneina (70,3 ± 11,2 %), respecto a la quercetina (63 ± 10,5 %) y al control (66,1 ± 11,2 %) (P < 0,05). Para MP, HA, VCL, VSL y VAP, el tratamiento control presentó valores superiores a los tratamientos con antioxidantes (P < 0,05). Se concluye que la ergotioneina y la quercetina incrementan la CAT e influyen sobre la movilidad y la cinética post-descongelación del semen equino.

The aim of this study was to evaluate the antioxidant capacity and post-thaw quality of stallion semen cryopreserved with quercetin and ergothioneine. Nine ejaculates from three Colombian Creole horses were cryopreserved under three treatments: ergothioneine (100 pM), quercetin (100 pM) and control (no antioxidant). Post-thaw were evaluated the parameters: total antioxidant capacity (TAC) of semen through the acid test of azino 2,2'-bis[3-ethylbenzothiazoline]-6-sulfonic acid (ABTS•+); total motility (MT), progressive motility (MP), hyperactivity (HA) and curvilinear (VCL), linear (VSL) and average path (VAP) velocities by the computerized system SCA ; structural membrane integrity and acrosome integrity by fluorescence microscopy using SYBR / IP and FITC / PNA probes, respectively; morphology by eosinnigrosin staining and functional membrane integrity by hypoosmotic swelling test (HOS). The adjustment of generalized linear models (GLM) and comparison of means by Tukey was performed. The TAC (pmol trolox/ml) of thawed semen was higher for ergothioneine (4.0 ± 0.3) and quercetin (3.9 ± 0.4), compared to control (2.6 ± 1.5). For MT a higher average for ergothioneine (70.3 ± 11.2%) compared to quercetin (63 ± 10.5%) and control (66.1 ± 11.2%) was found (P < 0.05). For MP, HA, VCL, VSL and VAP, the control showed higher values compared to the antioxidant treatments (P < 0.05). It is concluded that ergothioneine and quercetin increased the TAC and have influence on post-thawed motility and kinetics of stallion semen.

Differential abundances of four forms of Binder of SPerm 1 in the seminal plasma of Bos taurus indicus bulls with different patterns of semen freezability.

The Binder of SPerm 1 (BSP1) protein is involved in the fertilization and semen cryopreservation processes and is described to be both beneficial and detrimental to sperm. Previously, the relationship of BSP1 with freezability events has not been completely understood. The objective of this work was to determine the differential abundance of the forms of the BSP1 protein in cryopreserved seminal plasma of Bos taurus indicus bulls with different patterns of semen freezability using proteomics. A wide cohort of adult bulls with high genetic value from an artificial insemination center was used as donors of high quality, fresh semen. Nine bulls presenting different patterns of semen freezability were selected. Two-dimensional gel electrophoresis showed differential abundance in a group of seven protein spots in the frozen/thawed seminal plasma from the bulls, ranging from 15 to 17 kDa, with pI values from 4.6 to 5.8. Four of these spots were confirmed to be BSP1 using mass spectrometry, proteomics, biochemical, and computational analysis (Tukey's test at P < 0.05). The protein spot weighing 15.52 ± 0.53 kDa with a pI value of 5.78 ± 0.12 is highlighted by its high abundance in bulls with low semen freezability and its absence in bulls presenting high semen freezability. This is the first report showing that more than two forms of BSP1 are found in the seminal plasma of Nelore adult bulls and not all animals have a similar abundance of each BSP1 form. Different BSP1 forms may be involved in different events of fertilization and the cryopreservation process.

Strategies to improve the fertility of fresh and frozen donkey semen.

Fertility rates of donkey semen in jennies are lower compared to mares. The aims of this study were to evaluate different sperm cryopreservation methods and insemination strategies to improve the fertility of donkey semen in jennies. Three experiments were performed: (1) the comparison of two freezing methods of donkey semen (conventional method and automated method); (2) the determination of a suitable insemination dose of fresh donkey semen for jennies and mares; and (3) the influence of the semen deposition site on fertility of jennies inseminated with frozen donkey semen. For experiment 1, no differences were observed in total motility, angular velocity, curvilinear velocity, straight-line velocity, and plasma membrane integrity between samples frozen with the conventional (Styrofoam box) and the automated method (TK 4000C). However, the automated method provided higher values of progressive motility and rapid cells in frozen-thawed samples in comparison with the conventional method (P < 0.05). For experiment 2, mares were bred using 500 × 10(6) fresh sperm (M); and jennies using 1 × 10(9) (J1) or 500 × 10(6) fresh sperm (J5). Pregnancy rates in M, J1, and J5 were 93% (14/15), 73% (11/15), and 40% (6/15), respectively. When using different insemination doses, 500 × 10(6) or 1 × 10(9) sperm, no significant difference was observed in pregnancy rates of mares (M, 14/15) and jennies (J1, 11/15). Furthermore, there was no significant difference between the two insemination doses in jennies. However, with an insemination dose of 500 × 10(6) fresh sperm, the pregnancy rates were significantly higher in mares (M, 14/15) than in jennies (J5, 6/15; P < 0.05). For experiment 3, the inseminations were carried out in the uterine body (UB) or in the uterine horn of jennies with frozen-thawed donkey semen. No pregnancies were achieved with inseminations performed in the UB (0/12). The pregnancy rate for uterine horn group was 28.26% (13/46) and thus significantly higher than the UB group (0%; 0/12; P < 0.05). In conclusion, the automated method showed higher values on progressive motility and rapid cells parameters compared to the conventional method and can be used as an alternative for freezing donkey semen. The increase in the number of sperm cells per insemination dose using fresh donkey semen improved the fertility rates in jennies. The deep horn inseminations using frozen-thawed donkey semen increased the pregnancy rate in jennies, and the multiple inseminations may be an option to improve the fertility rates of donkey semen in jennies.

Técnicas de inseminação artificial em cães: revisão / Techniques of artificial insemination in dogs review / Las técnicas de inseminación artificial en los perros - revisión

La inseminación artificial en los perros es una herramienta de mejoramiento que aumenta la producción de 90% de la tasa de éxito de la fertilización. Es una aplica extensamente en los reproductores biotecnológicos, que consiste en la deposición del semen por medio artificial en el tracto reproductivo de la hembra. El semen puede ser manipulado en su forma fresca, refrigerada o criopreservados (congelados), y se utilizan diferentes técnicas de inseminación de acuerdo a su presentación. Para obtener la máxima eficacia de la técnica que se necesita principalmente un espécimen masculino, la correcta selección de animales adecuados para la reproducción, así como el diagnóstico de la época ideal, de acuerdo con el período fértil de la matriz. Para ello se llevan a cabo pruebas adicionales, como la citología vaginal, vaginoscopia y hormona de dosificación (o dosificación de progesterona). Dado que el método de inseminación artificial se utiliza correctamente, la inseminación es una herramienta útil para mejorar la calidad general de todas las razas, lo que permite uma gran mejora genética y la cría.(AU)

Artificial insemination in dogs is a breeding tool that increases production 90% of the fertilizationsuccess rate. It is a biotech widely applied in animal breeding, which consists of the deposition artificially semen in the reproductive tract of the female. Semen can be manipulated in its fresh form, cooled or cryopreserved (frozen), and used different insemination techniques according to your presentation. For most efficiency technique is primaríly needed a male specimen, the correct selection of animais suitable for breeding, as well as the optimal diagnosis point, according to the fertile period of the array. For this are performed additional tests such as cytology Vaginal, Vaginoscopy and Hormone dosing (or Progesterone dosage). Since the artificial insemination method is used correctly, the insemination is a useful tool to improve the overall quality of all races, allowing a large genetic and husbandry improvement.(AU)

A inseminação artificial em cães é uma ferramenta do melhoramento produtivo que aumenta até 90% a taxa de sucesso da fertilização. É uma biotécnica amplamente aplicada na reprodução animal, que consiste na deposição de forma artificial do sêmen no trato reprodutivo da fêmea. O sêmen pode ser manipulado na sua forma fresca, resfriada ou criopreservada (congelada), sendo utilizadas diferentes técnicas de inseminação de acordo com a sua apresentação. Para maior eficácia da técnica é necessário primeiramente um exemplar macho, a seleção correta de animais aptos à reprodução, assim como o diagnóstico do momento ideal, de acordo com o período fértil da matriz. Para isso são realizados exames complementares como: Citologia Vaginal, Vaginoscopia e Dosagem Hormonal (ou Dosagem de Progesterona). Desde que o método de inseminação artificial seja utilizado de forma correta, a inseminação é uma ferramenta útil para melhorar a qualidade geral de todas as raças, permitindo um grande aperfeiçoamento genético e zootécnico.(AU)

Técnicas de inseminação artificial em cães: revisão / Techniques of artificial insemination in dogs – review / Las técnicas de inseminación artificial en los perros - revisión

La inseminación artificial en los perros es una herramienta de mejoramiento que aumenta la producción de 90% de la tasa de éxito de la fertilización. Es una aplica extensamente en los reproductores biotecnológicos, que consiste en la deposición del semen por medio artificial en el tracto reproductivo de la hembra. El semen puede ser manipulado en su forma fresca, refrigerada o criopreservados (congelados), y se utilizan diferentes técnicas de inseminación de acuerdo a su presentación. Para obtener la máxima eficacia de la técnica que se necesita principalmente un espécimen masculino, la correcta selección de animales adecuados para la reproducción, así como el diagnóstico de la época ideal, de acuerdo con el período fértil de la matriz. Para ello se llevan a cabo pruebas adicionales, como la citología vaginal, vaginoscopia y hormona de dosificación (o dosificación de progesterona). Dado que el método de inseminación artificial se utiliza correctamente, la inseminación es una herramienta útil para mejorar la calidad general de todas las razas, lo que permite uma gran mejora genética y la cría.

Artificial insemination in dogs is a breeding tool that increases production 90% of the fertilizationsuccess rate. It is a biotech widely applied in animal breeding, which consists of the deposition artificially semen in the reproductive tract of the female. Semen can be manipulated in its fresh form, cooled or cryopreserved (frozen), and used different insemination techniques according to your presentation. For most efficiency technique is primaríly needed a male specimen, the correct selection of animais suitable for breeding, as well as the optimal diagnosis point, according to the fertile period of the array. For this are performed additional tests such as cytology Vaginal, Vaginoscopy and Hormone dosing (or Progesterone dosage). Since the artificial insemination method is used correctly, the insemination is a useful tool to improve the overall quality of all races, allowing a large genetic and husbandry improvement.

A inseminação artificial em cães é uma ferramenta do melhoramento produtivo que aumenta até 90% a taxa de sucesso da fertilização. É uma biotécnica amplamente aplicada na reprodução animal, que consiste na deposição de forma artificial do sêmen no trato reprodutivo da fêmea. O sêmen pode ser manipulado na sua forma fresca, resfriada ou criopreservada (congelada), sendo utilizadas diferentes técnicas de inseminação de acordo com a sua apresentação. Para maior eficácia da técnica é necessário primeiramente um exemplar macho, a seleção correta de animais aptos à reprodução, assim como o diagnóstico do momento ideal, de acordo com o período fértil da matriz. Para isso são realizados exames complementares como: Citologia Vaginal, Vaginoscopia e Dosagem Hormonal (ou Dosagem de Progesterona). Desde que o método de inseminação artificial seja utilizado de forma correta, a inseminação é uma ferramenta útil para melhorar a qualidade geral de todas as raças, permitindo um grande aperfeiçoamento genético e zootécnico.

Sperm characteristics following freezing in extenders supplemented with whole egg yolk and different concentrations of low-density lipoproteins in the collared peccary (Pecari tajacu).

The aim of the current study was to compare sperm quality characteristics of the collared peccary (Pecari tajacu) following freezing in extenders supplemented with whole egg yolk and different concentrations of low-density lipoproteins (LDL). Semen from 11 adult males was obtained by electroejaculation and evaluated for sperm motility, vigor, morphology as well as membrane integrity analyzed by the hypo-osmotic swelling (HOS) test and a fluorescent staining. Moreover, the semen was diluted in a Tris-based extender containing 20% egg yolk (control group) or 5, 10 or 20% LDL (treatment groups). The semen samples were frozen in liquid nitrogen and thawed in a water bath for 60s at 37°C. The treatments did not affect (p>0.05) sperm vigor, morphology or membrane integrity analyzed by the HOS test. However, post-thaw sperm motility was significantly higher (p<0.05) in the extender supplemented with 20% LDL (36.4 ± 5.3%) compared with the egg yolk extender and extender supplemented with 10% LDL. Furthermore, the percentage of membrane-intact frozen-thawed spermatozoa analyzed by the fluorescent staining was significantly higher (p<0.05) in the extender supplemented with 20% LDL (27.4 ± 6.5%) than in the other groups. In conclusion, 20% LDL can be used to substitute the whole egg yolk as a cryoprotective additive for freezing semen of the collared peccary.

Seminal plasma concentrations of Anti-Müllerian hormone and inhibin B predict motile sperm recovery from cryopreserved semen in asthenozoospermic men: a prospective cohort study.

The rate of motile sperm recovery after cryopreservation is very variable and difficult to predict. Anti-Müllerian hormone (AMH) and inhibin B are produced by Sertoli cells and released into the seminal plasma, where they could be functional markers of spermatogenesis and sperm resistance to thermal stress. The aim of this study was to evaluate whether seminal plasma levels of AMH and inhibin B predict sperm recovery after cryopreservation. The study included 153 men enrolled prospectively during a semen analysis. The cohort was stratified by the fresh semen characteristics into: normal (n = 52), high sperm count (n = 55), asthenozoospermia (n = 23), and oligozoospermia (n = 23). The main outcome measure was motile sperm recovery rate, defined as post-thaw total motile sperm count × 100/pre-freezing total motile sperm count. In men with asthenozoospermia there was a significant correlation between motile sperm recovery rate and the pre-freezing concentrations of AMH (r = 0.522, p < 0.05) and inhibin B (0.471, p < 0.05). In this group, the areas under the receiver operating characteristic curves of AMH and inhibin B for prediction of ≥50% motile sperm recovery after cryopreservation were, respectively, 0.808 and 0.638. AMH was particularly useful, with sensitivity of 0.85, specificity of 0.80, positive predictive value of 0.84 and negative predictive value of 0.80. The sensitivity, specificity, positive, and negative predictive values of inhibin B for the same outcome were, respectively, 0.62, 0.60, 0.67, and 0.55. The median motile sperm recovery rate was 83% when seminal plasma AMH concentration was ≥0.84 ng/mL, vs. 27% when AMH concentration was <0.84 ng/mL (p < 0.05). In other patient groups, there was no correlation between the two hormone levels in seminal plasma and the motile sperm recovery rate. In conclusion, seminal plasma AMH and inhibin B concentrations correlate with and can be used to predict motile sperm recovery after semen cryopreservation in asthenozoospermic men.

Viabilidade espermática do sêmen congelado de suínos da raça Piau avaliada pelo teste de termorresistência / Spermatic viability of cryopreserved semen of Piau swine breed analyzed by thermo resistant test

O objetivo do presente trabalho foi verificar o efeito de três protocolos de criopreservação de sêmen sobre a viabilidade espermática pós-descongelamento de suínos da raça Piau, avaliados por meio do teste de termorresistência (TTR). Para o congelamento, os ejaculados foram fracionados e submetidos aos seguintes tratamentos: protocolo 1 -preconizado por Fürst et al. (2005), modificado quanto aos meios diluentes; protocolo 2 -preconizado por Fürst et al. (2005), modificado quanto à curva de resfriamento; e protocolo 3 -preconizado por Ohata et al. (2001). Após o descongelamento o sêmen foi submetido ao teste de termo-resistência (38 °C por 2 horas), procedendo-se avaliações de motilidade e vigor espermáticos a cada 30 minutos. As médias registradas para motilidade imediatamente após o descongelamento foram de 20,9±12,4; 29,5±10,9 e 49,5±12,1%; respectivamente para os protocolos 1, 2 e 3. Os resultados do TTR evidenciam queda gradual dos parâmetros de MOT ao longo de 2 horas de duração nos três protocolos utilizados. Observou-se que os valores médios de motilidade espermática verificados no protocolo 3 foram maiores que as registradas nos outros dois protocolos, em todos os períodos avaliados. Do mesmo modo, a porcentagem média de espermatozoides móveis obtidos no protocolo 2 foi maior que a obtida no protocolo 1 nos primeiros 30 minutos de avaliação, sendo que após este período não houve diferença entre eles. Com relação ao vigor espermático entre os protocolos, verificou-se que o protocolo 3 apresentou resultados melhores em relação aos demais. As melhores médias para o protocolo 3, demonstram a superioridade do mesmo na criopreservação das células espermáticas dos animais do presente estudo.(AU)

The objective of this study was to verify three protocols of semen cryopreservation on spermatic viability after thawing from Piau swine breed, by thermo resistant test (TRT). To freezing, the ejaculates was split and submitted to three protocols: Protocol 1 -proposed by Furst et al. (2005), altered by diluent media; Protocol 2 -proposed by Furst et al. (2005), altered by cooled curve; and Protocol 3 -proposed by Ohata et al. (2001). After thawing, semen was submitted to TRT (37°C during 2 hours), and sperm motility was analyzed at 30 minutes of interval. Motility after thawing was 20.9±12.4, 29.5±10.9 and 49.5±12.1%, respectively to P1, P2 and P3. The TRT results show gradually decrease of motility and vigor along 2 hours of test procedure utilized. It was observed that the mean values of sperm motility observed in protocol 3 were higher than those recorded in the other two protocols in all periods. Similarly, the mean percentage of motile spermatozoa obtained in protocol 2 was higher than that obtained in protocol 1 in the first 30 minutes of evaluation, and after this period there was no difference between them. Regarding the sperm vigor between protocols, it was found that the protocol 3 showed better results than the other. The protocol 3 tested by Piau boars show highest values in cellular semen cryopreservation.(AU)

Viabilidade espermática do sêmen congelado de suínos da raça Piau avaliada pelo teste de termorresistência / Spermatic viability of cryopreserved semen of Piau swine breed analyzed by thermo resistant test

O objetivo do presente trabalho foi verificar o efeito de três protocolos de criopreservação de sêmen sobre a viabilidade espermática pós-descongelamento de suínos da raça Piau, avaliados por meio do teste de termorresistência (TTR). Para o congelamento, os ejaculados foram fracionados e submetidos aos seguintes tratamentos: protocolo 1 -preconizado por Fürst et al. (2005), modificado quanto aos meios diluentes; protocolo 2 -preconizado por Fürst et al. (2005), modificado quanto à curva de resfriamento; e protocolo 3 -preconizado por Ohata et al. (2001). Após o descongelamento o sêmen foi submetido ao teste de termo-resistência (38 °C por 2 horas), procedendo-se avaliações de motilidade e vigor espermáticos a cada 30 minutos. As médias registradas para motilidade imediatamente após o descongelamento foram de 20,9±12,4; 29,5±10,9 e 49,5±12,1%; respectivamente para os protocolos 1, 2 e 3. Os resultados do TTR evidenciam queda gradual dos parâmetros de MOT ao longo de 2 horas de duração nos três protocolos utilizados. Observou-se que os valores médios de motilidade espermática verificados no protocolo 3 foram maiores que as registradas nos outros dois protocolos, em todos os períodos avaliados. Do mesmo modo, a porcentagem média de espermatozoides móveis obtidos no protocolo 2 foi maior que a obtida no protocolo 1 nos primeiros 30 minutos de avaliação, sendo que após este período não houve diferença entre eles. Com relação ao vigor espermático entre os protocolos, verificou-se que o protocolo 3 apresentou resultados melhores em relação aos demais. As melhores médias para o protocolo 3, demonstram a superioridade do mesmo na criopreservação das células espermáticas dos animais do presente estudo.

The objective of this study was to verify three protocols of semen cryopreservation on spermatic viability after thawing from Piau swine breed, by thermo resistant test (TRT). To freezing, the ejaculates was split and submitted to three protocols: Protocol 1 -proposed by Furst et al. (2005), altered by diluent media; Protocol 2 -proposed by Furst et al. (2005), altered by cooled curve; and Protocol 3 -proposed by Ohata et al. (2001). After thawing, semen was submitted to TRT (37°C during 2 hours), and sperm motility was analyzed at 30 minutes of interval. Motility after thawing was 20.9±12.4, 29.5±10.9 and 49.5±12.1%, respectively to P1, P2 and P3. The TRT results show gradually decrease of motility and vigor along 2 hours of test procedure utilized. It was observed that the mean values of sperm motility observed in protocol 3 were higher than those recorded in the other two protocols in all periods. Similarly, the mean percentage of motile spermatozoa obtained in protocol 2 was higher than that obtained in protocol 1 in the first 30 minutes of evaluation, and after this period there was no difference between them. Regarding the sperm vigor between protocols, it was found that the protocol 3 showed better results than the other. The protocol 3 tested by Piau boars show highest values in cellular semen cryopreservation.

Efeito da glutationa reduzida (GSH) na criopreservação de espermatozóides da espécie canina: avaliação in vitro e in vivo / Effect of reduced glutathione (GSH) in the cryopreservation of canine spermatozoa: in vitro and in vivo evaluation

O presente experimento teve por objetivos comparar a adição de diferentes concentrações de glutationa reduzida (GSH) no diluidor para criopreservação do sêmen de cães, considerando-se as características morfofuncionais pós-descongelação; e verificar os índices de gestação e número de filhotes por ninhada obtidos a partir da inseminação artificial intra-uterina, por cateterização cervical endoscópica, com sêmen criopreservado contendo diferentes concentrações de glutationa. Na primeira fase do experimento, foram realizadas duas colheitas seminais de 11 reprodutores com intervalo mínimo de uma semana entre os procedimentos. Foram empregadas três concentrações diferentes de glutationa reduzida (0, 10 e 20 mM) ao meio de congelação tris-gema-citrato. O sêmen fresco, refrigerado, glicerolizado e descongelado foram avaliados por citometria de fluxo com uso de sondas específicas para determinar a integridade das membranas plasmática e acrossomal, potencial de membrana mitocondrial e fragmentação de DNA. Também foi realizada dosagem de substâncias reativas ao ácido tiobarbitúrico (TBARS) para avaliar os níveis de estresse oxidativo. Na segunda fase do experimento, foram inseminadas 6 fêmeas, alocadas em dois grupos: inseminação intra-uterina com sêmen congelado em diluidor contendo 10 mM (IA-GSH10, n=3) e sem a adição de glutationa (IA-GSH0, n=3). O acompanhamento do ciclo estral foi realizado por citologia vaginal, vaginoscopia, dosagem de progesterona e LH. Foram realizadas duas inseminações no 5o e 6o dias após pico de LH e a gestação diagnosticada aos 30 dias. A adição de 10 e 20 mM de GSH ao diluidor para criopreservação promoveu queda na motilidade espermática nas amostras descongeladas, sendo o maior prejuízo observado na concentração de 20 mM. Foi detectada maior porcentagem de acrossomos íntegros nos grupos tratados. A glicerolização e, principalmente, a exposição do espermatozóide ao nitrogênio líquido e posterior descongelação foram responsáveis pela diminuição da motilidade espermática, em consequência ao prejuízo da função mitocondrial promovida pela produção de radicais livres durante o processamento seminal. A gestação foi diagnosticada em duas fêmeas do grupo IA-GSH10 e duas do grupo IA-GSH0. Em conclusão, a adição de 10 e 20 mM de GSH ao diluidor para criopreservação seminal não promoveu os efeitos protetores esperados na espécie canina, sendo a concentração de 20 mM responsável por maiores prejuízos à amostra seminal. A adição de 10 mM de GSH ao diluidor para criopreservação seminal na espécie canina manteve o índice de fertilidade semelhante ao obtido com sêmen criopreservado sem a suplementação antioxidante

The present experiment aimed to compare the addition of different concentrations of reduced glutathione (GSH) on frozen-thawed canine semen, considering the morphofunctional characteristics; and to verify the pregnancy rate and number of puppies per litter obtained from intrauterine insemination by endoscopic catheterization of the cervix, with frozen-thawed semen containing different concentrations of glutathione. During the first phase of the experiment, two seminal samples collected weekly from 11 mature dogs were used. Three different concentrations of reduced glutathione (0, 10 and 20 mM) were supplemented in a tris-citrate egg yolk extender. The fresh, chilled, glycerolized and post-thawed semen were assessed by flow cytometry using specific probes to determine plasma membrane and acrosomal integrity, mitochondrial membrane potential and DNA fragmentation. Thiobarbituric acid reactive substances (TBARS) assay was also performed to assess the occurrence of oxidative stress. In the second phase of the experiment, six canine females were allocated into two groups: intrauterine insemination with frozen-thawed semen containing 10 mM GSH (IA-GSH10, n = 3) and without the addition of glutathione (IA-GSH0, n = 3). Bitches were monitored by vaginal cytology, vaginoscopy, progesterone and LH assays. Two inseminations were performed on days 5 and 6 post LH peak. We verified that the addition of 10 and 20 mM of GSH to semen extender promoted a decrease in post-thaw sperm motility, as well as a higher damage degree at the 20 mM concentration. We also detected a higher percentage of acrossomal integrity in treated groups. At glycerolization and moreover, the sperm exposure to liquid nitrogen and further thawing, were responsible for the decrease in sperm motility, due to the loss of mitochondrial function through the free radicals production. Gestation was diagnosed in two female from IA-GSH10 group and two bitches of the IA-GSH0 group. In conclusion, we did not observe the expected protective effects of the addition of 10 and 20 mM GSH to semen extender. Furthermore, the concentration of 20 mM was responsible for the more extreme damage to semen sample. The supplementation of 10 mM GSH to semen extender for cryopreservation maintained the fertility rates similar to the ones observed for cryopreservation without antioxidant supplementation in dogs