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Submitted by Vitor Silverio Rodrigues (vitorsrodrigues@reitoria.unesp.br) on 2014-05-27T11:30:39Z No. of bitstreams: 0Bitstream added on 2014-05-27T14:37:58Z : No. of bitstreams: 1 2-s2.0-84883189464.pdf: 403114 bytes, checksum: fc18e5299501abbc1714c79e55e8740a (MD5) Made available in DSpace on 2014-05-27T11:30:39Z (GMT). No. of bitstreams: 0 Previous issue date: 2013-09-03 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) After a serious injury or sudden death, epididymis cauda sperm recovery and cryopreservation may present as the last opportunity to obtain genetic material from a valuable stallion. This study evaluated the viability of cooled equine sperm collected by three different methods: sperm of ejaculated (G1), sperm recovered from the epididymal cauda immediately after orchiectomy (G2) and sperm recovered from the epididymal cauda after storage for 24 hours at 5°C (G3). To obtain G1 sperm, two ejaculates were collected. After 1 week, all stallions underwent a bilateral orchiectomy, and one of the removed epididymides was flushed to obtain G2 sperm. The contralateral epididymis was stored at 5°C for 24 hours before being flushed to obtain G3 sperm. The sperm samples were evaluated immediately after the addition of the refrigeration extender, and after 24 and 48 hours of storage at 5°C. After 24 and 48 hours of storage, the epididymal sperm demonstrated higher motility traits when compared to the ejaculated sperm (P

Submitted by Vitor Silverio Rodrigues (vitorsrodrigues@reitoria.unesp.br) on 2014-05-27T11:30:51Z No. of bitstreams: 0 Made available in DSpace on 2014-05-27T11:30:51Z (GMT). No. of bitstreams: 0 Previous issue date: 2013-10-15 Cryopreservation of spermatozoa is a pivotal tool in assisted reproduction, and studies aiming to establish optimal freezing/thawing protocols are essential to enhance sperm survival. The objectives of the present study were to (1) compare the cryoprotective efficiency of three different glycerol concentrations (3%, 5%, and 7%) on the basis of post-thaw sperm quality and (2) investigate whether the incidence of morphologically abnormal sperm in fresh samples is related to cryodamage sensitivity. Semen was collected from six tomcats using an artificial vagina (total 18 ejaculates). Each ejaculate was diluted using Tris-egg yolk-based extender (TEY), evaluated, equally divided into three aliquots, and rediluted using TEY with and without glycerol to achieve final concentrations of 3%, 5%, and 7%. Samples were loaded into 0.25 mL straws, equilibrated for 60 minutes at 5 °C, frozen, and then thawed at 46 °C for 12 seconds. Fresh and frozen-thawed samples were evaluated for sperm motion parameters (computer-assisted sperm analysis), plasma membrane integrity (PMI; propidium iodide and carboxyfluorescein diacetate), and DNA integrity (acridine orange). Plasma and acrosomal membrane integrity were assessed by flow cytometry (propidium iodide and fluorescein isothiocyanate-conjugated pea (Pisum sativum) agglutinin) immediately after thawing. Sperm motion parameters were also evaluated at 30 and 60 minutes of postincubation. For all treatment groups, cryopreservation significantly impaired the PMI and sperm motion parameters, except for straightness and amplitude of lateral head displacement. DNA integrity showed a slight reduction (P < 0.05) when 3% glycerol was used. The percentage of total motility, progressive motility, and rapid spermatozoa were significantly lower immediately after thawing and up to 60 minutes of incubation for the 3% glycerol group when compared with 5% and 7%. No difference (P > 0.05) was found for PMI, acrosome integrity, and DNA integrity among post-thaw groups. However, higher (P < 0.05) incidence of viable cells with reacted acrosome and dead cells with intact acrosome were observed with 7% and 3% glycerol, respectively. Percentage of morphologically abnormal spermatozoa in fresh sample was positively correlated with PMI only in the 3% glycerol group and negatively correlated with sperm motility in the 5% and 7% groups. In conclusion, the final concentration of 5% glycerol offered better cryoprotective effect for ejaculated cat sperm, and the relationship found between prefreezing sperm morphology and post-thaw sperm quality showed to be dependent on final glycerol concentration. © 2013 Elsevier Inc. Department of Animal Reproduction and Veterinary Radiology, FMVZ São Paulo State University, Botucatu, São Paulo Laboratory of Flow Cytometry Fundação Amaral Carvalho, Jaú, São Paulo Department of Biostatistics Institute of Biosciences (IBB) São Paulo State University, Botucatu, São Paulo Department of Animal Reproduction and Veterinary Radiology, FMVZ São Paulo State University, Botucatu, São Paulo Department of Biostatistics Institute of Biosciences (IBB) São Paulo State University, Botucatu, São Paulo

Made available in DSpace on 2016-07-07T12:37:41Z (GMT). No. of bitstreams: 0 Previous issue date: 2013. Added 1 bitstream(s) on 2016-07-07T12:46:23Z : No. of bitstreams: 1 ISSN0100-2430-2013-35-04-306-310.pdf: 381575 bytes, checksum: b572082c4d33c8a54678c675b919ef00 (MD5) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Na espécie equina a utilização do sê- men congelado tem fatores limitantes como a necessidade de mão de obra especializada, o alto custo dos equipamentos, assim como locais apropriados nos haras para manipulação do sêmen. Com o objetivo de avaliar o efeito do plasma seminal na refrigeração do sêmen equino por 12 horas antes da criopreservação, o estudo foi conduzido com cinco garanhões da raça Mangalarga Marchador, com idade entre 4 e 8 anos. Foram realizadas 15 colheitas de sêmen, sendo que cada ejaculado foi divido em três grupos, e cada grupo foi dividido em dois subgrupos, utilizando diferentes meios extensores (Botu-Sêmen® e Botu-Turbo®). A amostra do Grupo I (controle) foi congelada logo após a coleta, o sêmen do Grupo II teve o plasma seminal mantido durante a refrigeração e o sêmen do Grupo III teve o plasma seminal retirado para refrigerar. Após o descongelamento, os parâmetros espermáticos de motilidade foram analisados utilizando CASA e a integridade da membrana plasmática foi avaliada por meio de sondas fluorescentes de diacetato de carboxifluoresceína e iodeto de propídio. A aná- lise estatística foi realizada através da ANOVA e teste de Tukey. Não houve diferença significativa (P>0,05) entre o sêmen, previamente a congelação, refrigerado com e sem plasma seminal para todos os parâmetros espermáticos avaliados, sendo estes motilidade total (50,5 vs 50,6), motilidade progressiva (21,3 vs 24,1) e integridade de membrana plasmática (26,2 vs 28). Os dados analisados indicaram que a refrigeração do sêmen equino por 12 horas antes da criopreservação é um método eficaz, que possibilita um melhor aproveitamento do ejaculado, independentemente da manutenção ou remoção do plasma seminal durante a refrigeração. In the equine species, the use of frozen semen has limiting factors as the need for skilled labor, the high cost of equipment, as well as an appropriate place on the farms to the handling of semen. In order to evaluate the effect of seminal plasma on cooling of equine semen for 12 hours before cryopreservation, this study was conducted with five stallions of Mangalarga Marchador breed, aged between 4 and 8 years old. For this experiment, 15 semen collections were performed, each ejaculate was divided into three groups and each group was divided into two subgroups, using different media extenders (Botu-Semen® and Botu-Turbo®). In Group I (control), the semen was frozen immediately after collection; in Group II, the seminal plasma was maintained during the cooling and in Group III, the seminal plasma was removed before cooling. After thawing, the sperm parameters were analyzed using CASA and membrane integrity was evaluated by the fluorescent probes carboxyfluorescein diacetate and propidium iodide. Statistical analysis was performed by ANOVA and Tukey’s test. There was no significant differences (P>0.05) between the semen samples with and without seminal plasma prior to freezing for all sperm parameters evaluated, total motility (50.5 vs 50.6), progressive motility (21.3 vs 24.1) and plasma membrane integrity (26.2 vs 28). The analyzed data showed that the cooling of equine semen for 12 hours before cryopreservation is an effective method that enables a better use of the ejaculate, regardless of maintenance or removal of seminal plasma during the semen storage. Universidade Estadual Paulista Júlio de Mesquita Filho (UNESP), Faculdade de Medicina Veterinária e Zootecnia (FMVZ), Departamento de Reprodução Animal e Radiologia Veterinária, Botucatu, SP, Brasil Universidade Federal Rural do Rio de Janeiro (UFRRJ), Departamento de Reprodução e Avaliação Animal, Seropédica, RJ, Brasil Universidade Estadual Paulista Júlio de Mesquita Filho (UNESP), Faculdade de Medicina Veterinária e Zootecnia (FMVZ), Departamento de Reprodução Animal e Radiologia Veterinária, Botucatu, SP, Brasil

Submitted by Vitor Silverio Rodrigues (vitorsrodrigues@reitoria.unesp.br) on 2014-05-27T11:26:27Z No. of bitstreams: 0 Made available in DSpace on 2014-05-27T11:26:27Z (GMT). No. of bitstreams: 0 Previous issue date: 2012-04-07 Semen cryopreservation is still considered suboptimal due to lower fertility when compared to fresh semen. The reasons for the loss of fertility are various and related to irreversible damage caused to the cells during the freeze-thaw process. An alternative to conventional cryopreservation represents the use of chilled bull semen, preventing the damage associated with freezing, thereby guaranteeing greater sperm viability. The aim of this study was to describe the use of cooled bull semen as a strategy to increase the pregnancy for Fixed-Time Artificial Insemination (FTAI) of Nellore (Bos indicus) cows. One ejaculate of a select Nellore bull obtained by electroejaculation was used; the semen sample was fractioned into two aliquots: one diluted in Botu-Bov® extender containing 6.4% glycerol for cryopreservation (BB-F, frozen group) and one diluted in the same extender, free from cryoprotectants and used for cooling (BB-C, cooled semen group). The samples in the BB-C group were chilled to 5°C using an isothermic box and maintained for 24 h prior to use. A total of 349 lactating Nellore cows (70-90 days after birth) were synchronized by the insertion of a progesterone releasing device (1.0 g) and estradiol benzoate (2.0 mg i.m.) on a random day of the estrous cycle (Day 0); FTAI was performed 44-48 h after the removal of the device. The pregnancy rates were 45.71 and 61.49% (P

The objective of this study was to investigate reproductive characteristics of stallions at a tropical zone in the breeding and non-breeding seasons. The following parameters were assessed: testicular volume; semen quality; and serum concentrations of LH, FSH, and testosterone; in addition to the percentages of germ cells and proportions of germ cells/Sertoli cells by testicular cytology in stallions. Semen was collected from eight adult stallions twice a week during a 12-week period in both seasons (6 weeks before and 6 weeks after the summer and winter solstices). Jugular blood samples were collected periodically for hormone analysis by radioimmunoassay during the same periods. Testicular measures and cytological samples were taken at the end of each period. Mean concentration of testosterone was significantly higher (P = 0.04) during the breeding season and the proportion of Sertoli cells/100 germ cells in cytological smears was significantly lower during the breeding season (P = 0.0001). Effects of season were not significant either for testicular volume or for any semen parameter (P > 0.05). Seasonal changes in the mean concentrations of LH and FSH were not observed (P > 0.05). There were also no significant differences in the mean percentages of germ cell types between both seasons (P > 0.05). Lack of seasonal differences in the testicular volume and semen parameters of tropical stallions are probably due to the small variation in duration of natural light between the observed periods, slightly under 3 h.

Os corantes Panótico Rápido e Giemsa são frequentemente utilizados para a avaliação de células de linhagem espermatogênica, porém nenhuma destas colorações foi desenvolvida especificamente para o processamento tintorial desse tipo celular. Como até presente não existe consenso na literatura sobre qual das técnicas de coloração permite a melhor visualização das células germinativas testiculares, o objetivo deste estudo foi determinar a melhor coloração para esfregaços obtidos por punção biópsia aspirativa testicular (PBA) de bovinos. Foram selecionados 25 machos bovinos adultos, que sofreram PBAs em ambos os testículos (n=50 punções biópsia aspirativas, perfazendo um total de 100 esfregaços testiculares). O conteúdo de cada PBA foi utilizado para a confecção de no mínimo 2 esfregaços em lâminas foscas de vidro que posteriormente foram corados utilizando dois métodos distintos: Panótico Rápido (G1, n=50 esfregaços) ou Giemsa (G2, n=50). Para avaliação da qualidade dos esfregaços as células da linhagem espermatogênica foram contabilizadas e comparadas entre os dois grupos experimentais. A facilidade e nitidez de visualização proporcionada por cada uma das técnicas foi comparada atribuindo-se uma nota de 1 a 3 (1: ruim; 2: regular; 3: boa). A coloração Panótico Rápido permitiu a identificação de maior quantidade de espermatócitos secundários (p=0,0042) e células inflamatórias (p=0,0318), apresentando ainda efeito significativo sobre a qualidade de leitura dos esfregaços (p=0,0087). Embora ambas as colorações permitam a identificação e visualização das estruturas da linhagem espermatogênica, o Panótico Rápido permitiu melhor visualização e avaliação das células testiculares.

The cryopreservation of epididymal sperm is important to preserve genetic material from valuable deceased males. This study evaluated the viability of sperm samples from eight stallions under three conditions: (1) collected using an artificial vagina (EJ-0h), (2) recovered from the epididymal cauda immediately after orchiectomy (EP-0h), and (3) recovered from the epididymal cauda after 24h of storage at 5°C (EP-24h). To obtain EJ-0h sperm, two ejaculates were collected from each stallion. After 1 week, the stallions were submitted to bilateral orchiectomy, and one of the removed epididymides was flushed to obtain EP-0h sperm. The contralateral epididymis was stored at 5°C for 24h before being flushed to obtain EP-24h sperm. The sperm samples were analyzed at three different times: immediately after sperm recovery, after dilution in the freezing extender, and post-thawing. A fertility trial was performed using 39 estrous cycles. After ovulation induction with 1mg of deslorelin acetate (i.m.), mares were inseminated with 800×10(6) sperm. The total number of sperm recovered was 7.8±4.7×10(9) for EJ-0h sperm, 12.9±9.2×10(9) for EP-0h sperm and 12.0±8.0×10(9) for EP-24h sperm. The sperm motility, evaluated by total motility, progressive motility and the percentage of rapid cells, was similar among the samples before and after freezing (P0.05). However, the plasma membrane integrity was different between EJ-0h and EP-0h pre-freezing and between EJ-0h and EP-24h post-thawing (P0.05). The conception rates were similar between groups inseminated with sperm recovered from the epididymal cauda immediately after orchiectomy (EP-0h), after 24h of storage at 5°C of the epididymal cauda (EP-24h) and with ejaculated sperm (EJ-0h) (P0.05). In conclusion, the viability and fertility of cauda epididymal sperm are similar to those of ejaculated sperm.

Made available in DSpace on 2016-04-01T18:44:14Z (GMT). No. of bitstreams: 0 Previous issue date: 2010. Added 1 bitstream(s) on 2016-04-01T18:49:46Z : No. of bitstreams: 1 ISSN0378-4320-2010-121-01-186-187.pdf: 85775 bytes, checksum: ac2d45d3d4ee8bd33ac1c9b26f7c4e4f (MD5) Universidade Estadual Paulista Júlio de Mesquita Filho, Departamento de Cirurgia Veterinária e Reprodução Animal, Faculdade de Medicina Veterinária e Zootecnia de Botucatu, Botucatu, UNESP- Campus de Botucatu- Rubião Jr-, Rubião Jr, CEP 18618000, SP, Brasil Universidade Estadual Paulista Júlio de Mesquita Filho, Departamento de Cirurgia Veterinária e Reprodução Animal, Faculdade de Medicina Veterinária e Zootecnia de Botucatu, Botucatu, UNESP- Campus de Botucatu- Rubião Jr-, Rubião Jr, CEP 18618000, SP, Brasil

Made available in DSpace on 2016-04-01T18:44:14Z (GMT). No. of bitstreams: 0 Previous issue date: 2010. Added 1 bitstream(s) on 2016-04-01T18:49:47Z : No. of bitstreams: 1 ISSN0378-4320-2010-121-01-178-179.pdf: 96576 bytes, checksum: 8fb40716f73b8e12f798baf47cfcb93c (MD5) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Universidade Estadual Paulista Júlio de Mesquita Filho, Departamento de Cirurgia Veterinária e Reprodução Animal, Faculdade de Medicina Veterinária e Zootecnia de Botucatu, Botucatu, UNESP- Campus de Botucatu- Rubião Jr-, Rubião Jr, CEP 18618000, SP, Brasil Universidade Estadual Paulista Júlio de Mesquita Filho, Departamento de Cirurgia Veterinária e Reprodução Animal, Faculdade de Medicina Veterinária e Zootecnia de Botucatu, Botucatu, UNESP- Campus de Botucatu- Rubião Jr-, Rubião Jr, CEP 18618000, SP, Brasil FAPESP: 2008/57504-2

Made available in DSpace on 2016-04-01T18:44:21Z (GMT). No. of bitstreams: 0 Previous issue date: 2010. Added 1 bitstream(s) on 2016-04-01T18:49:51Z : No. of bitstreams: 1 ISSN0378-4320-2010-121-01-168-170.pdf: 593173 bytes, checksum: e693ecec394342ab838a2765096e715a (MD5) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Universidade Estadual Paulista Júlio de Mesquita Filho, Departamento de Reprodução Animal e Radiologia Veterinária, Botucatu, FMVZ-UNESP-Botucatu-SP, Rubião Junior, CEP 18618-000, SP, Brasil Universidade Estadual Paulista Júlio de Mesquita Filho, DDepartamento de Clínica Veterinária, Botucatu, FMVZ-UNESP-Botucatu-SP, Rubião Junior, CEP 18618-000, SP, Brasil Universidade Estadual Paulista Júlio de Mesquita Filho, Departamento de Reprodução Animal e Radiologia Veterinária, Botucatu, FMVZ-UNESP-Botucatu-SP, Rubião Junior, CEP 18618-000, SP, Brasil Universidade Estadual Paulista Júlio de Mesquita Filho, DDepartamento de Clínica Veterinária, Botucatu, FMVZ-UNESP-Botucatu-SP, Rubião Junior, CEP 18618-000, SP, Brasil

This study was aimed at assessing the changes in sperm motion patterns and the percentage of acrosome reaction (AR) in domestic cat semen after treatment with either ionomycin or progesterone (P(4)). Ten ejaculates were collected from five tomcats using an artificial vagina, and were diluted, centrifuged and resuspended in a capacitation medium. Samples were evaluated and divided into seven equal aliquots and, after 2 h at 25 degrees C, were incubated for 30 min at 38 degrees C in 5% CO(2) and then analyzed. Computer-assisted sperm analysis and a combination of three fluorescent probes were used to assess sperm plasma, acrosomal membrane integrity and mitochondrial transmembrane potential. Thirty minutes after the start of incubation, P(4) was added (10 microg/ml) to the P1 group. Groups P2 and P3 were supplemented with P(4) (10 and 20 microg/ml, respectively) only after 2 h of incubation, and groups I1 and I2 were supplemented with ionomycin (4 and 8 mum, respectively) 2 h after incubation. Group E was supplemented with ethanol (0.6%) at 2 h after incubation and group C received no supplementation. Ionomycin and P(4) treatments led to a hyperactivation-like sperm motion and an increase (p0.05) in the percentage of AR. Although a higher (p0.05) percentage of AR was obtained in group I2 when compared with all P(4) groups, a decrease (p0.05) in total and progressive motility was observed in I2 group. As I1 group was similar to I2 to induce AR without diminishing sperm motility, we can conclude that ionomycin at 4 microm seems to be more suitable to trigger AR in domestic cat sperm.

The aim of this study was to compare the efficiency of the intravaginal (IVAI) vs. intrauterine artificial insemination (IUAI) using frozen-thawed sperm in the domestic cat. Semen was collected from two tom cats using an artificial vagina and samples were assessed for motility (computer-assisted sperm analysis (CASA)), sperm morphology and plasma membrane integrity. After dilution with TRIS/OEP/YOLK (4% of glycerol), sperm samples were loaded into 0.25 mL straws (25 x 10(6)motile sperm/straw), incubated at 5 degrees C for 20 min and cryopreserved over liquid nitrogen (LN(2)) vapor for 15 min and then immersed in LN(2). For each AI, four straws from the same male were thawed (12s at 46 degrees C) and centrifuged at 250 x g for 8 min to pellet the sperm. The supernatant was discarded and sperm pellet resuspended with the remaining liquid, approximately 100 microL, and analyzed as described above. Queens were treated with a single im injection of 100 IU eCG to induce ovarian follicular development. Final oocyte maturation and ovulation was induced with 100 IU hCG given im at 82-84 h after eCG administration. Thirty hours after hCG administration, females were inseminated either intrauterine (n=8 queens) or intravaginally (n=8 queens), using thawed sperm from a single male. Although a pronounced decrease in sperm motility, acrosome and plasma membrane integrity was observed in sperm samples from both cats, a pregnancy rate of 75% was achieved when using the intrauterine AI method compared with 0% pregnancy when inseminated intravaginally.

Submitted by Guilherme Lemeszenski (guilherme@nead.unesp.br) on 2013-08-22T18:59:45Z No. of bitstreams: 1 S0103-84782006000500025.pdf: 341614 bytes, checksum: 088ffeab321138d9f044c57a966ac317 (MD5) Made available in DSpace on 2013-08-22T18:59:45Z (GMT). No. of bitstreams: 1 S0103-84782006000500025.pdf: 341614 bytes, checksum: 088ffeab321138d9f044c57a966ac317 (MD5) Previous issue date: 2006-10-01 Made available in DSpace on 2013-09-30T19:57:02Z (GMT). No. of bitstreams: 2 S0103-84782006000500025.pdf: 341614 bytes, checksum: 088ffeab321138d9f044c57a966ac317 (MD5) S0103-84782006000500025.pdf.txt: 17011 bytes, checksum: b48f3728c151c9372a20356f5b7740a1 (MD5) Previous issue date: 2006-10-01 Submitted by Vitor Silverio Rodrigues (vitorsrodrigues@reitoria.unesp.br) on 2014-05-20T15:14:49Z No. of bitstreams: 2 S0103-84782006000500025.pdf: 341614 bytes, checksum: 088ffeab321138d9f044c57a966ac317 (MD5) S0103-84782006000500025.pdf.txt: 17011 bytes, checksum: b48f3728c151c9372a20356f5b7740a1 (MD5) Made available in DSpace on 2014-05-20T15:14:49Z (GMT). No. of bitstreams: 2 S0103-84782006000500025.pdf: 341614 bytes, checksum: 088ffeab321138d9f044c57a966ac317 (MD5) S0103-84782006000500025.pdf.txt: 17011 bytes, checksum: b48f3728c151c9372a20356f5b7740a1 (MD5) Previous issue date: 2006-10-01 Descreve-se um caso de infertilidade de um jumento SRD confirmada por meio de microscopia eletrônica de transmissão (MET). O espermiograma, avaliado sob microscopia ótica, revelou baixa motilidade e alta concentração de anormalidades espermáticas do tipo gota citoplasmática proximal. O material foi avaliado por MET, observando-se um acúmulo desordenado de microtúbulos causando protusões irregulares na região do colo espermático. O último teste realizado correspondeu ao de fertilidade in vivo, utilizando-se quatro éguas portadoras de bom histórico reprodutivo, nas quais não foi possível confirmar nenhuma prenhez. Frente aos resultados obtidos, associados aos achados da MET, estabeleceu-se o diagnóstico de infertilidade associada a defeito microtubular dos espermatozóides. A donkey infertility was described by transmission electron microscopy (MET). The spermiogram evaluated by light microscopy showed low sperm motility and high concentration of abnormal sperm with proximal cytoplasmic droplets. The material was evaluated by MET, where it was observed disarrangement of microtubules, causing irregular protrusions in the spermatic neck. The last test done was the in vivo fertility, using four mares, with a reproductive healthy historic, where no pregnancy occurred. Facing the results that we had in vivo, associated with the MET findings, we diagnosed infertility associated with microtubular defect of the spermatozoa. Universidade Estadual Paulista Júlio de Mesquita Filho Faculdade de Medicina Veterinária e Zootecnia Universidade Estadual Paulista Júlio de Mesquita Filho Faculdade de Medicina Veterinária e Zootecnia

Stallion semen cryopreservation, despite its impact on the horse industry, is not an established technology. During the last years, a number of modifications have been proposed to the freezing process, however, a large population of stallions still have poor semen quality and fertility after frozen-thawed. Glycerol toxicity could be a reason for the variation on stallion sperm freezability. There are limited publications concerning the use of alternative cryoprotectants for equine sperm. Glycerol is contraceptive for some species and other cryoprotectors, such as amides, have been show to be a good option for freezing semen of these species. Recent reports have shown encouraging data respecting the use of amides as cryoprotectants for stallions, with more remarkable improvements for semen from stallions that freeze poorly when glycerol is used.

Summary Early pregnancy factor (EPF) appears to be a protein complex that depends on pregnancy. It has been detected in the serum of several animal species using the Rosette Inhibition Test. In the present study 30 adult, Mangalarga mares with normal reproductive activity were divided into two groups: an experimental group which were mated and/or inseminated, and an unmated control group. In the experimental group, blood samples were taken on the 7th, 14th and 21st days after ovulation. In the control group, blood samples were collected from seven mares in oestrus and eight in dioestrus. In the control group the Rosette Inhibition Titre (RIT) ranged from 3 to 8 × 103, whereas in the experimental group a RIT higher than 8 × 103 was considered an indication of the presence of EPF. Serum progesterone concentrations on Day 7 were equal to or greater than those measured in the mated mares on Day 14 or Day 21.

Universidade Estadual Paulista Julio de Mesquita Filho, Departamento de Cirurgia Veterinaria e Reproducao Animal, Faculdade de Medicina Veterinaria e Zootecnia de Botucatu, Botucatu, UNESP- Campus de Botucatu- Rubiao Jr-, Rubiao Jr, CEP 18618000, SP, Brasil

Daily ultrasound examinations were conducted from Days 10 to 60 (ovulation = Day 0) of pregnancy to monitor the conceptus in jennies (n = 12). The embryonic vesicle was first detected on Day 11.5 +/- 0.9 (mean +/- SD; range 10 to 13 d) and was mobile until movement ceased (fixation) on Day 15.5 +/- 1.4 (range, 13 to 18 d). The vesicle was spherical from Days 10 to 18 (mean growth rate, 3.2 mm/d), non spherical (irregular) with a reduced growth rate (0.5 mm/d) from Days 19 to 29, and then grew at a moderate rate (1.6 mm/d) up to Day 46. On average, detection of the embryo proper (consistently located on the ventral aspect of the yolk sac) and embryonic heartbeat were Days 20.7 +/- 1.2 and 23.5 +/- 1.3, respectively. Formation of the allantoic sac was first detected on Day 24.4 +/- 1.7 and was complete on Day 36.8 +/- 1.6. Descent of the fetus (and formation of the umbilical cord) began on Day 37.9 +/- 1.7 and was complete on Day 44.1 +/- 2.1. Crown-rump length averaged 3.7, 15.4, 22.7, 37.5 and 59.6 mm on Days 20, 30, 40, 50 and 60, respectively. In general, morphologic features and dates of occurrence were similar to those reported previously in the mare.

Sao Paulo State Univ, Dept Anim Reprod & Vet Radiol, FMVZ UNESP, BR-18618000 Botucatu, SP, Brazil

Ovarian follicular activity was studied by ultrasonography during 17 oestrous cycles in 9 Mangalarga mares during the second half of the ovulatory season. Sixteen oestrous cycles were considered normal and one 3-wave cycle showing a prolonged luteal phase was considered atypical. Daily ultrasonographic examinations were performed and the compiled data on follicular dynamics were studied retrospectively. One major wave of follicular growth was observed in 13 of the 16 normal cycles (81.25%), whereas 2 major waves occurred in 3 cycles (18.75%). The mean (+/- s.d.) days of emergence of the primary wave of follicular development in cycles containing one or 2 waves were Day 6.0 +/- 2.3 and Day 11.0 +/- 1.0, respectively. The secondary wave of follicular development in 2-wave cycles emerged on Day 0.0 +/- 3.6. The day of wave divergence for primary waves of follicular development in cycles which exhibited one or 2 major waves were Day 12.2 +/- 3.5 and Day 17.3 +/- 3.0, respectively. Divergence of secondary waves occurred in only one of the 3 cycles which exhibited 2 major follicular waves (Day 7). The mean (+/- s.d.) maximum diameters of the dominant follicle in the primary wave of oestrous cycles exhibiting one and 2 major waves were 39.0 +/- 3.9 mm and 34.7 +/- 2.5 mm, respectively. The mean (+/- s.d.) maximum diameter of the dominant follicle present in the secondary wave was 34.3 +/- 11.0 mm. The mean (+/- s.d.) lengths of the interovulatory intervals for cycles containing one and 2 major waves were 19.4 +/- 2.2 and 23.3 +/- 2.5 days, respectively. These data indicate that most Mangalarga mares show one major follicular wave during the oestrous cycle but a small percentage of mares show 2 major waves.

Submitted by Guilherme Lemeszenski (guilherme@nead.unesp.br) on 2013-08-22T19:04:05Z No. of bitstreams: 1 S1413-95961998000400005.pdf: 556898 bytes, checksum: c866722681b5ca5d0cd4c5fede2f6578 (MD5) Made available in DSpace on 2013-08-22T19:04:05Z (GMT). No. of bitstreams: 1 S1413-95961998000400005.pdf: 556898 bytes, checksum: c866722681b5ca5d0cd4c5fede2f6578 (MD5) Previous issue date: 1998-01-01 Made available in DSpace on 2013-09-30T20:06:40Z (GMT). No. of bitstreams: 2 S1413-95961998000400005.pdf: 556898 bytes, checksum: c866722681b5ca5d0cd4c5fede2f6578 (MD5) S1413-95961998000400005.pdf.txt: 15280 bytes, checksum: 0ae551a8b7a55d6951385de3cef3f23a (MD5) Previous issue date: 1998-01-01 Submitted by Vitor Silverio Rodrigues (vitorsrodrigues@reitoria.unesp.br) on 2014-05-20T13:41:52Z No. of bitstreams: 2 S1413-95961998000400005.pdf: 556898 bytes, checksum: c866722681b5ca5d0cd4c5fede2f6578 (MD5) S1413-95961998000400005.pdf.txt: 15280 bytes, checksum: 0ae551a8b7a55d6951385de3cef3f23a (MD5) Made available in DSpace on 2014-05-20T13:41:52Z (GMT). No. of bitstreams: 2 S1413-95961998000400005.pdf: 556898 bytes, checksum: c866722681b5ca5d0cd4c5fede2f6578 (MD5) S1413-95961998000400005.pdf.txt: 15280 bytes, checksum: 0ae551a8b7a55d6951385de3cef3f23a (MD5) Previous issue date: 1998-01-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) O presente estudo objetivou diagnosticar as causas da morte embrionária precoce em 128 éguas. Amostras de soro foram coletadas nos dias 4, 7, 10, 13, 16, 19, 21 e 30 após ovulação, para dosagens hormonais e mensurações do corpo lúteo. O diâmetro e as características da vesícula embrionária foram avaliados, através da ultra-sonografia, a partir do 12º dia pós-ovulação. Das 128 éguas estudadas, 17 (13.28%) apresentaram morte embrionária. O diâmetro do corpo lúteo, bem como as concentrações plasmáticas de progesterona, foi semelhante nos grupos que apresentaram morte embrionária e nos que mantiveram a gestação. Os níveis de estrogeno plasmático foram mais elevados no grupo das fêmeas que mantiveram a gestação. Os exames citológicos, microbiológicos e histopatológicos revelaram que a maioria das éguas com diagnóstico de morte embrionária eram portadoras de endometrites. The present study aimed to diagnose early embryonic death in 128 mares. Blood samples were collected at the 4th, 7th, 10th, 13th, 16th, 19th, 21st and 30th day after ovulation and the diameter of the corpus luteum was measured. The diameter and characteristics of the embryonic vesicle were evaluated by ultrasonography. Microbiology, cytology and histopathology were carried out in mares with embryonic death. Plasma oestrogen and progesterone concentration were measured by radioimmunoassay. From 128 mares, 17 (13.28%) showed early embryonic death. The embryonic losses took place at 19 days of pregnancy in 47.05% and at 21 days of pregnancy in 29.4% of the mares. The diameter of the corpus luteum in the mares that maintained pregnancy was similar to those with embryonic loss. Otherwise, the diameter of the embryonic vesicles was bigger at the 16th, 19th, 21st and 30th day of pregnancy when compared to the mares with embryonic loss. The mean plasma progesterone concentration was similar in both groups and the median plasma oestrogen concentration was higher in the pregnant mares. The cytological, microbiological and histopathological exams revealed that most of the mares had endometritis. Ultrasonography provided an early diagnosis of pregnancy (from the 12th day post-ovulation) and important information about the development of embryo and embryonic death. Endometritis was considered the main cause of embryonic losses in this study. Universidade Estadual Paulista Faculdade de Medicina Veterinária e Zootecnia Departamento de Reprodução Animal e Radiologia Veterinária Universidade Estadual Paulista Faculdade de Odontologia Departamento de Clínica, Cirurgia e Reprodução Animal Universidade Estadual Paulista Faculdade de Medicina Veterinária e Zootecnia Departamento de Higiene Veterinária e Saúde Pública Universidade Estadual Paulista Faculdade de Medicina Veterinária e Zootecnia Departamento de Reprodução Animal e Radiologia Veterinária Universidade Estadual Paulista Faculdade de Odontologia Departamento de Clínica, Cirurgia e Reprodução Animal Universidade Estadual Paulista Faculdade de Medicina Veterinária e Zootecnia Departamento de Higiene Veterinária e Saúde Pública

Seminal plasma removal, an indispensable step in equine semen cryopreservation, is usually done by centrifugation, but this might cause mechanical damage to sperm. A new method for seminal plasma removal from stallion semen, namely a filter composed of a synthetic hydrophilic membrane (Sperm Filter, BotuPharma, Botucatu, Sao Paulo, Brazil), was recently proposed. The objective of this study was to test the use of the Sperm Filter in the removal of seminal plasma before freezing stallion semen. Ejaculates from 31 stallions were divided into two groups and cryopreserved. In group 1 (G1), seminal plasma was removed with the Sperm Filter, and in group 2 (G2), seminal plasma was removed by centrifugation (600× g for 10 minutes). There were no differences (P < 0.05) between G1 and G2 in sperm kinetic parameters or plasma membrane integrity before or after cryopreservation. However, sperm recovery rate was higher (P < 0.05) for G1 versus G2 (mean ± SD, 89.4 ± 7.4% vs. 80.9 ± 5.5%). Therefore, the Sperm Filter was as efficient as centrifugation in removing seminal plasma from the stallion ejaculate. However, filtering was more practical and had significantly fewer sperm lost than the centrifugation technique.

The aim of this study was to evaluate the generation of hydrogen peroxide (H2O2) and membrane lipid peroxidation of subfertile spermatozoa stored at 5°C for 24 h. Semen samples, collected from 5 subfertile stallions (≤40% of conception rate), were diluted in a skim-based extender (BotuSemen; Botupharma) and stored in a passive transport container at 5°C over a period of 24 h. Sperm motility was determined by computer-assisted semen analysis (CASA; IVOS 12, Hamilton Thorne Inc., Beverly, MA, USA) for total motility (TM), progressive motility (PM), and rapid sperm (RAP). Plasma membrane integrity, generation of H2O2, and membrane lipid peroxidation were determined by flow cytometry (LSRFortessa cell analyzer, BD Biosciences, Franklin Lakes, NJ, USA). For evaluation of acrosome and plasma membrane integrity, samples were stained with Hoechst 33342 dye (H33342; Molecular Probes, Eugene, OR, USA), iodide propidium (IP; Sigma, St. Louis, MO, USA), and fluorescein isothiocyanate-conjugated Pisum sativum agglutinin (FITC-PSA; Sigma). For the assessment of generation of H2O2, 2′,7′-dichlorofluorescein diacetate (DCFH-DA; Sigma), H33342, and IP were added to the sperm suspension. For the assessment of sperm lipid peroxidation, samples were stained with C11-BODIPY581/591 (Molecular Probes), H33342, and IP. Samples were evaluated after semen collection (0 h) and after 24 h of cooled storage. A total of 10 000 gated events were analyzed per sample by flow cytometry. The green fluorescence (FL1) was collected through a 580-nm band-pass filter and the red fluorescence (FL3) through a 635-nm band-pass filter. Statistical analysis was performed using GraphPad Prism version 4.03 (2005; GraphPad Software Inc., La Jolla, CA, USA), through paired t-test to identify the significant differences (P ≤ 0.05). In general, sperm motility parameters of TM and RAP, and acrosome and plasma membrane integrity significantly decreased after 24 h of refrigeration at 5°C (P ≤ 0.05). Interestingly, no differences were found in PM at 0 and 24 h (P ≥ 0.05). The percentage of sperm cells with high generation of H2O2 did not differ at 0 and 24 h (P ≥ 0.05), whereas the percentage of sperm cells with membrane peroxidation increased (0.56 ± 0.3 v. 2.24 ± 1.3; P ≤ 0.05) at these periods. The percentage of viable sperm cells with low generation of H2O2 had a significant decrease from 22.6 ± 11.2 at 0 h to 0.08 ± 0.1 after 24 h of storage (P ≤ 0.05), although no differences were found in the percentage of viable sperm cells without membrane peroxidation (P ≥ 0.05). In conclusion, the cooled storage of subfertile spermatozoa for 24 h drastically decreased the number of viable spermatozoa with low generation of H2O2 and increased the percentage of membrane lipid peroxidation, which is related to the decrease in sperm motility and increase in dead sperm. These results make it difficult to use refrigerated semen of subfertile stallions with poor semen quality in commercial breeding programs. São Paulo Research Foundation (FAPESP) is acknowledged for supporting this research.

Artificial insemination with cooled-shipped semen has been widely used in horse breeding. However, some stallions, referred to as poor coolers, present abrupt fertility decrease when their semen is processed, cooled, and transported. Cholesterol incorporation into sperm membranes improves the quality of cryopreserved semen by increasing the sperm membrane stability and fluidity at low temperatures. Despite the beneficial effect of cholesterol addition on sperm quality, studies demonstrate that the presence of large amounts of cholesterol in the plasma membrane interferes with the physiological process of sperm capacitation and is detrimental to frozen equine sperm fertility. The aim of this study was to assess the fertility of cooled semen from good-cooler and poor-cooler stallions after adding cholesterol to sperm membranes. Two stallions were used and classified as good cooler (n = 1) and poor cooler (n = 1) based on the ability to maintain sperm progressive motility after 24 h of cooling at 5°C. For classification of the stallions, the fertility history was also taken into account through the results of pregnancy per cycle using inseminations with cooled semen (70% for good cooler). Ejaculates of these stallions were subjected to 2 treatments: control (CON) and cholesterol (CLC). In the CON group, the semen was extended to 30 × 106 sperms mL–1 with skim milk-based extender (BotuSemen™). In the CLC group, the semen was extended as in the CON group plus 0.25 µL/1 × 106 sperms of 6.1 mM cholesterol-loaded cyclodextrin was added. Afterwards, both treatments were cooled at 5°C for 24 h. To test the fertility of poor-cooler and good-cooler stallions, 2 cycles from 25 mares and 2 cycles from 10 mares were respectively used. For both stallions, randomly for each mare, the inseminations were performed by alternating both treatments. If the mare was first inseminated with the CLC treatment, in the next cycle the CON treatment was used and vice versa. After 24 h of ovulation induction, the inseminations were done in the uterine body with 1 × 109 viable cells. Statistical analyses were performed using the Fisher exact test and significance was set at P

Alterations to a sperm cell that are similar to apoptosis reduce the sperm cell longevity in the female reproductive tract. The aim of the present study was to evaluate the effect of sperm motility factors [caffeine, heparin, and penicillamine, hypotaurine, and epinephrine (PHE)] on phosphatidylserine translocation in equine epididymal semen. Equine epididymal samples were obtained from 10 stallions using retrograde flush with 40 mL of BotuSemen® (BS) per epididymal cauda. The samples were divided into 3 groups: BS (control), Fert-TALP (Fert), and Sperm-TALP (SP), and diluted 1 : 1 with these media. Samples containing 100 × 106 sperm cells were loaded into straws, and then incubated at room temperature (25°C) for 15 min. The samples were centrifuged (1000g for 10 min), the supernatant was removed and the pellets were resuspended in BotuCrio®. Samples were loaded into 0.5-mL straws and cooled at 5°C for 20 min. Then, they were frozen in nitrogen vapor (6 cm above the level of LN) for an additional 20 min and finally plunged into nitrogen and stored. The straws were thawed at 46°C/20 min. Sperm motility was evaluated by computer-assisted semen analysis (CASA; HTM-IVOS 12). To evaluate the pancreatic secretory trypsin inhibitor (PSTI), the Annexin V-FITC Apoptosis Detection Kit (6710KK; Pharmingen, San Jose, CA, USA) was used according to the manufacturer’s recommendations. The samples were diluted in Annexin V buffer solution to a concentration of 2 × 106 spermatozoa mL–1 and then 5 mL of Annexin V-FITC, 5 mL of propidium iodide (PI, 50 mg mL–1), and 2 mL of Hoechst 33342 dye (H342, 40 mg mL–1) were added. The samples were then homogenized and incubated for 15 min. Then, 400 µL of Annexin V buffer solution was added to obtain a final concentration of 1 × 106 spermatozoa mL–1. The samples were evaluated. Flow cytometry was carried out at BD LSR II (Becton Dickinson, Mountain View, CA, USA). Mean and standard deviation were calculated, and then the normality test was evaluated by the Kolmogorov-Smirnov test. An analysis of variance and Tukey test with a P

Previous studies in humans and current studies in horses show that the presence of leukocytes in the semen promotes increased reactive oxygen species and proinflammatory cytokines production. These substances cause damage to membrane integrity, mitochondrial function, DNA integrity, and lead to increased expression of apoptosis markers in the spermatic cell. As a result of these effects, decreased quality and longevity of the semen that lead to fertility reduction could be highlighted. A 6-year-old stallion was admitted in the Department of Animal Reproduction and Veterinary Radiology of Veterinary Medicine and Animal Science School, UNESP, Botucatu-SP, Brazil, with a history of pyospermia due to seminal vesiculitis. Therefore, because of the high demand of the stallion semen and attempting to allow its use in equine embryo transfer programs, a process to reduce leukocyte numbers was performed. For the evaluation of the percentage of leukocytes in the sample, a 10-µL aliquot of semen was smeared and stained with Quick PanoticTM. Semen was diluted in skim milk-based extender BotuSemenTM (1 : 1; Nidacon International AB, Mölndal, Sweden) and then analyzed for sperm kinetic parameters by computer assisted semen analysis (CASA). Sequentially, seminal plasma was removed by filtration with SpermFilterTM (Nidacon International AB). The sample was resuspended with BotuSemenTM to a concentration of 300 × 106 sperm mL–1 and subjected to a single layer gradient centrifugation (EquiPureTM; Nidacon International AB) at 400g for 20 min. A proportion of 5 mL of diluted semen in 5 mL of EquiPureTM (1 : 1) was used for the centrifugation. The pellet was resuspended with BotuSemenTM at a concentration of 50 × 106 sperm mL–1. Before and after processing, sperm kinetic means were, respectively: Total motility (TM) = 46% and 89%, progressive motility (PM) = 20% and 55%, and rapid sperm cells (RAP) = 32% and 81%. The mean percentages of leukocytes in semen smears before and after processing was 80% and 5%, respectively. The processed semen was used for artificial insemination of 5 mares and none of them showed signs of post-breeding endometritis. In addition, in all cases, embryos were recovered when uterine flushes were done on Day 8 post-ovulation. In conclusion, the method used for the processing of semen with pyospermia was effective in reducing leukocyte loads and promoted an improvement of seminal kinetics parameters which allowed its use in embryo transfer programs. The authors acknowledge FAPESP (2011/23139-9) for financial support and Botupharma for donation of BotuSêmenTM extender and SpermFilterTM.

The ultrarapid freezing technique was developed as an alternative to slow conventional freezing to avoid formation of ice crystals (Tucker MJ and Liebermann J 2007, Vitrification in Assisted Reproduction, 87-92). The recent use of ethylene glycol tetraacetic acid (EGTA) instead of glycerol as a cryoprotectant for sperm is a result of efforts to reduce osmotic and cytotoxic effects. Accordingly, the objective of the present study was to compare the cryoprotective effects of EGTA v. glycerol on bovine sperm frozen using an ultrarapid method. A pool of epididymal sperm collected from 5 bulls was maintained in Botusui extender (Biotech, Botucatu, Brazil) containing 5% BSA, either EGTA (5%) or glycerol (5%), and 0 or 2 mg mL of polyvinyl alcohol (PVA). All samples were loaded into 0.25-mL French straws and either placed into a freezing machine (TK 4000 compacta, TK equipamentos para reproducao, Uberaba, Brazil) and cooled at a rate of 50°C/min, or exposed to liquid nitrogen (LN2) vapor for 5 to 15 min before plunging into LN2. After overnight storage in LN2, straws were thawed and examined using CASA (HTM IVOS 12, Hamilton Thorne Research, Beverly, MA, USA) for total motility (TM), progressive motility (PM), path velocity (VAP), progressive velocity (VSL), and track speed (VCL). Acridine Orange (AO) was used to check DNA integrity of sperm cells (Chirinea VH et al. 2006 Ciencia Animal Brasileira 7, 407-415). Our results indicate that, despite the potentially damaging effects of the extreme temperature changes, epididymal sperm showed 95% of DNA integrity and, therefore, should be able to participate in the fertilization process. Addition of PVA had a negative effect on sperm motility characteristics. CASA revealed that glycerol was a more suitable cryoprotectant for ultrarapid freezing of bull epididymal spermatozoa than was EGTA (see Table 1). Table 1. CAPES.

The aim of the present study was to analyze the use of in vitro fertilization to predict the field fertility of Nelore bulls by using statistical models based on Bayesian inference. A total of 407 oocytes were obtained from slaughterhouse ovaries and submitted to in vitro maturation in TCM-199 and fertilization in Tyrode’s + BSA + lactate + pyruvate. Sperm cells from 3 different bulls, namely, V, T, and G, were used. All bulls had a known field gestational rate. After 18 h of IVF, a sample of zygotes (n = 40/bull) was stripped and stained with acetic orcein (1%) to estimate the number of pronuclei. The remaining zygotes (n = 287) were cultured in SOF for 7 to 8 days. The results are based on 3 replicates with 32 to 33 oocytes/bull per replicate. All 3 bulls analyzed presented similar percentages of oocytes with at least 2 pronuclei (52.5, 40, and 40% for bulls V, T, and G, respectively). However, when cleavage rate was analyzed, bull T presented a significantly (P = 0.0022) lower rate (57.4%) compared with bulls V (77.9%) and G (78.1%). In contrast, blastocyst formation rates were similar between bulls (27.9, 22.8, and 30.5% for bulls V, T, and G, respectively). When the cleavage and blastocyst rates were analyzed in comparison with the field pregnancy rates previously obtained for bulls V, T, and G by using statistic models based on Bayesian inference, the predictions of conception rate based on cleavage were 59.8 ± 2.5%, 54.2 ± 4.7%, and 59.8 ± 2.5% for bulls V, T, and G, respectively. When blastocyst formation rates were used, the predicted conception rate was 58.5 ± 2.2%, 52.3 ± 4.5%, and 61.5 ± 2.8% for bulls V, T, and G, respectively. The predicted conception rates obtained by using the cleavage and blastocyst data together were 54.7 ± 3.9%, 54.1 ± 4.8%, and 63.0 ± 3.1% for bulls V, T, and G, respectively, which was very similar to the real field conception rates for the same bulls (54.4, 54.1, and 63.3%, respectively). The statistical model obtained through the use of Bayesian inference with the combined data for cleavage and blastocyst rates presented a good correlation with the real field fertility of bulls used in this experiment. This result indicates that this statistical method could be a good and efficient alternative to predict the fertility of Nelore bulls. However, a larger number of animals will need to be tested by the same methodology to confirm these results. FAPESP.

The possibility of harvesting stallion epididymal spermatozoa from excised testis represents an important tool for preserving valuable genetic material from males that die unexpectedly, need to be euthanized or castrated. Freezing epididymal sperm after cooling is essential to guarantee the preservation of this valuable genetic material. Equine epididymal spermatozoa have been proven to be fertile; however, recent attempts achieving pregnancies have been frustrating. Due to a lack of information concerning the storage capacity of epididymis and total number of spermatozoa that can be obtained from the epididymal tail, the aim of the present study was to correlate testicular volume (length×width× height) and testicular weight with the recovery of epididymal spermatozoa retrieval. Testes were obtained during routine castration from twenty-two Brazilian jumping horses aging approximately 3 years. The animals were bred in the same farm and maintained under similar conditions. Testicular pair from the same animal was identified and all testes were stored without direct contact to ice in a thermic box cooled at 5 ◦C. After 24 h of cooling, the testes were washed with Ringer lactate solution aiming to remove blood and other contaminants. The epididymides were dissected and testes were weighted individually. The epididymal tails from the same stallion were flushed with 30 mL of a skim milk based extender (Botu-Semen®) using a 20L pipette tip attached to a 2-mL syringe. Final volume was measured and sperm concentration assessed. Mean volume recovered from both epididymides after flushing through the deferent duct was 51.14± 7.55 mL. The average concentration of recovered spermatozoa was 103.59± 78.17× 106 sperm/mL. This way, the mean number of recovered spermatozoa from a pair of epididymides was 4.88± 3.29× 109. Comparisons were made by Pearson correlation analysis, with p< 0.05 taken as significant. Positive correlations were found for weight× volume (r= 0.93), weight× sperm concentration (r= 0.59) and volume× sperm concentration (r= 0.61). Comparing both testes from the same animal, we observed a correlation between left and right testicular volume (r= 0.52) and weight (r= 0.65). The low correlation found between left and right testes may be due to the fact that five animals from the 22 horses used in this study presented great differences for volume and weight concerning the testicular pair. Perhaps the use of young stallions that could be in various degrees of sexual maturity may have contributed to the lower correlations when comparing the same animal. We can conclude that, among healthy horses, testicular volume and weight are correlated with sperm production and epididymal sperm retrieval.

Use of frozen semen by the horse industry is popular. Several extenders have been used to freeze stallion semen; the most used are EDTA-lactose egg yolk and INRA 82. A new extender named Botu-Crio� (BioTech, Botucatu, Sao Paulo, Brazil) has been used on a large scale in Brazil. No comparisons of these extenders have been published. The present experiment was designed to compare the ability of these three extenders to preserve sperm longevity after incubation. One ejaculate from each of 13 stallions was used. After collection using an artificial vagina, semen was filtered and diluted 1:1 with a milk-base extender (Botu-Semen�), split into 3 parts, and then centrifuged (600g/10 min). After removal of the supernatants, each semen pellet was resuspended with an aliquot of one of the extenders (INRA 82, EDTA-Lactose, or Botu-Crio) to a final concentration of 100 � 106 sperm cells mL–1. Semen was packaged in 0.5-mL French straws, and cooled for stabilization for 20 min at 5�C for Botu-Crio (BC), for 2 h at 5�C for INRA 82 (IR), and without stabilization for EDTA-Lactose (EL) extender. After stabilization, the straws were placed in nitrogen vapor for 15 min and then plunged into liquid nitrogen. Before motility evaluation by CASA using the Hamilton System Analyzer (Hamilton Thorne Biosciences, Beverly, MA, USA), straws were thawed (46�C/20 s) and then incubated at 37�C in a dry block. The patterns of total motility (TM) and progressive motility (PM) were evaluated immediately after thawing (T0), and at 10 (T10), 30 (T30), and 60 (T60) min after incubation at 37�C. Statistical analysis was performed using ANOVA and Tukey's test. Total motility, respectively, for IR, EL, and BC was TM0: 10%, 35%, and 57%; TM10: 25%, 37%, and 68%; TM30: 20%, 21%, and 56%; and TM60: 13%, 10%, and 41%. The respective progressive motility was PM0: 3%, 16%, and 28%; PM10: 11%, 16%, and 32%; PM30: 10%, 8%, and 26%; and PM60: 5%, 2%, and 16%. Motilities were always superior (P < 0.05) at all incubation times with the utilization of Botu-Crio extender. A fertility trial comparing the three extenders is in progress.

Em 52 éguas de diferentes raças e idades, praticou-se cirurgia corretiva de dilaceraçâo perineal de 3º grau, com modificações na técnica de GÖTZE, na tentativa de restabelecer a anatomia do sistema reprodutor, bem como a vida reprodutiva destas fêmeas. Das éguas que sofreram a cirurgia, a totalidade (100%) retornou à reprodução, alcançando-se o índice máximo de fertilidade após inseminação artificial ou cobertura. Os resultados obtidos pelo emprego da técnica proposta permite indicá-la como uma conduta correta e eficiente na recuperação anátomo-funcional do genital de éguas com dilaceraçâo de períneo de 3º grau. Corrective surgery of the 3rd degree perineal laceration was peformed in 52 mares of different breeds and ages, employing modified GÖTZE procedure to restore the normal anatomy of the reproductive system, as well as the normal reproductive condition of the females. All operated mares returned to reproductive activity and conceived after artificial insemination or natural mating. The results obtained showed that the practice of the modified technique would be a correct and effectice procedure for anatomical and functional recuperation of the genital system of mares with third degree perineal laceration.

Made available in DSpace on 2019-10-04T12:32:09Z (GMT). No. of bitstreams: 0 Previous issue date: 2018-09-01. Added 1 bitstream(s) on 2019-10-09T18:33:38Z : No. of bitstreams: 1 S0100-736X2018000901726.pdf: 800883 bytes, checksum: cecbc5035225453f4c7de006c441eb91 (MD5) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) To date, no studies have been performed evaluating the effect of boar spermatozoa concentration in 0.5mL freezing straws, leading us to examine this question. Each sperm-rich fraction of the ejaculate (n=25) was diluted at five different sperm concentrations (100, 200, 300, 600 and 800 x 10(6) spermatozoa/mL), packaged in 0.5mL straws, and subsequently frozen. After thawing, the sperm from all of treatment groups were analyzed to determine motility characteristics using a sperm class analyzer (SCA-CASA), and their plasma and acrosomal membrane integrity, mitochondria! membrane potential, sperm membrane lipid peroxidation and fluidity were analyzed by flow cytometry. An increase in spermatozoa concentration above 300x10(6) spermatozoa/mL in a 0.5mL straw impaired (p0.05) by high spermatozoa concentrations at freezing. Therefore, to increase spermatozoa survival and total and progressive motility after thawing, boar spermatozoa should be frozen at concentrations up to 300x10(6) spermatozoa/mL. Univ Sao Paulo, Fac Med Vet & Zootecnia, Nucl Pesquisa Suinos, BR-13635900 Pirassununga, SP, Brazil Univ Estadual Paulista, Fac Med Vet & Zootecnia, Dept Reprod Anim & Radiol Vet, UNESP, BR-18618970 Botucatu, SP, Brazil Univ Estadual Paulista, Fac Med Vet & Zootecnia, Dept Reprod Anim & Radiol Vet, UNESP, BR-18618970 Botucatu, SP, Brazil FAPESP: 2011/23484-8

Submitted by Vitor Silverio Rodrigues (vitorsrodrigues@reitoria.unesp.br) on 2014-05-27T11:28:34Z No. of bitstreams: 0 Made available in DSpace on 2014-05-27T11:28:34Z (GMT). No. of bitstreams: 0 Previous issue date: 2013-03-01 In the present study, different freezing systems (Styrofoam box and Mini Digitcool ZH 400) and storage volumes (0.5- and 0.25-mL straws) were compared with regard to sperm kinetics and plasma membrane integrity of frozen and thawed semen. For that, three ejaculates from four animals were frozen in Styrofoam box and Mini Digitcool ZH 400 machine. The 0.5-mL straws were thawed at 46°C for 20 seconds, and the 0.25-mL straws were thawed at 46°C for 12 seconds. Statistical analysis was performed using program R of descriptive analysis box plot, followed by analysis of variance using PROC MIXED of SAS 9.1 package. Variances of 5% were considered as different. There was no interaction between the straw sizes and volumes; however, statistical differences were observed between the semen storage volumes. The 0.5-mL straws had higher total motility (%), progressive motility (%), average path velocity (μm/s), straight-line velocity (μm/s), curvilinear velocity (μm/s), and rapid sperm percentage (%) than the 0.25-mL straws. However, plasma membrane integrity analysis did not differ between the two straws. Thus, it is possible to conclude that equine sperm cryopreserved in 0.5-mL straws has better sperm kinetics than when stored in 0.25-mL straws. Additionally, it is possible to conclude that automated systems that enable faster freezing rates result in a seminal quality that is similar to the one obtained by the conventional system using Styrofoam boxes. © 2013 Elsevier Inc. Department of Animal Reproduction and Veterinary Radiology School of Veterinary Medicine and Animal Science UNESP, Botucatu Department of Animal Reproduction and Veterinary Radiology School of Veterinary Medicine and Animal Science UNESP, Botucatu

Made available in DSpace on 2013-09-27T14:54:13Z (GMT). No. of bitstreams: 1 WOS000309793700001.pdf: 187330 bytes, checksum: 5ed93be917f7240ac370aef1fa74e928 (MD5) Previous issue date: 2012-11-01 Made available in DSpace on 2013-09-30T18:25:28Z (GMT). No. of bitstreams: 1 WOS000309793700001.pdf: 187330 bytes, checksum: 5ed93be917f7240ac370aef1fa74e928 (MD5) Previous issue date: 2012-11-01 Submitted by Vitor Silverio Rodrigues (vitorsrodrigues@reitoria.unesp.br) on 2014-05-20T13:41:58Z No. of bitstreams: 1 WOS000309793700001.pdf: 187330 bytes, checksum: 5ed93be917f7240ac370aef1fa74e928 (MD5) Made available in DSpace on 2014-05-20T13:41:58Z (GMT). No. of bitstreams: 1 WOS000309793700001.pdf: 187330 bytes, checksum: 5ed93be917f7240ac370aef1fa74e928 (MD5) Previous issue date: 2012-11-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) One of the main factors known to influence quality and fertility of bovine cryopreserved semen is the extender used. In this matter, a great worldwide interest has been directed to the development of chemically defined media, free of animal origin products. The objective of the present study was to compare the efficacy of three bovine semen extenders: Iris-fructose (IRIS, control with 20% egg yolk), Botu-Bov (R) (BB; 20% egg yolk), and Botu-Bov (R)-soy lecithin (BB-L; 1% soy lecithin). Towards this aim, post-thaw computer assisted sperm analysis (CASA), sperm membrane and acrosome integrity were evaluated (Experiment 1). Additionally, cryopreserved samples were used in a fixed time artificial insemination program aiming to evaluate in vivo fertility (pregnancy per insemination-P/AI; Experiment 2). Despite the higher straightness and linearity found for samples cryopreserved in BB and BB-L when compared to those cryopreserved in IRIS, egg yolk based extenders provided higher total and progressive motilities, percentage of rapid sperms and intact membrane cells (P < 0.05). Furthermore, P/IA was higher in samples cryopreserved in egg yolk based extenders when compared to soy lecithin [TRIS=59.26(a) (64/108), BB=62.37(a) (58/93), and BB-L=36.45(b) (35/96)]. Although soy lecithin represents an alternative for the development of chemically defined extenders with decreased risk of biological contamination, egg yolk based extenders are more efficient on the preservation of bovine post-thaw sperm viability and fertility. (C) 2012 Elsevier B.V. All rights reserved. São Paulo State Univ, Dept Anim Reprod & Vet Radiol, Botucatu, SP, Brazil Univ Santo Amaro, UNISA, Sch Vet Med, São Paulo, Brazil Univ São Paulo, Dept Anim Reprod, São Paulo, Brazil São Paulo State Univ, Dept Clin Surg & Anim Reprod, Aracatuba, SP, Brazil São Paulo State Univ, Dept Anim Reprod & Vet Radiol, Botucatu, SP, Brazil São Paulo State Univ, Dept Clin Surg & Anim Reprod, Aracatuba, SP, Brazil FAPESP: 06/61153-5

Submitted by Vitor Silverio Rodrigues (vitorsrodrigues@reitoria.unesp.br) on 2014-05-27T11:28:34Z No. of bitstreams: 0 Made available in DSpace on 2014-05-27T11:28:34Z (GMT). No. of bitstreams: 0 Previous issue date: 2013-03-01 The recovery of sperm from the epididymal cauda may be the last chance to obtain genetic material when sudden death or serious injuries occur in valuable stallions. However, the lack of technical knowledge regarding the storage and transportation of the epididymis often prevents the preservation of the sperm. Therefore, the aim of this study was to compare sperm parameters of sperm obtained immediately after orchiectomy with sperm recovered from epididymal cauda at different times after storage at 5°C and at room temperature (RT). For that, 48 stallions of different breeds were used. In group 1 (control group), eight stallions were used, and the harvest of the epididymal sperm was performed immediately after orchiectomy. In group 2, 40 stallions were used, which were divided into five groups according to the storage time of the epididymis after orchiectomy (6, 12, 18, 24, or 30 hours), making a total of eight stallions per group. One epididymis of each stallion was stored at 5°C, and the contralateral epididymis was stored at RT, both for the same period. The sperm parameters of total motility, progressive motility, progressive linear velocity, curvilinear velocity, percentage of rapid sperm, and plasma membrane integrity were evaluated in all the groups after sperm recovery, resuspension in a sperm freezing diluent, and thawing. In conclusion, the storage of the testis-epididymis complex at 5°C provided better preservation of epididymal sperm than the storage at RT, and regardless of the temperature, the progressive motility is the sperm parameter that is most sensitive to storage time. © 2013 Elsevier Inc. Department of Animal Reproduction and Veterinary Radiology School of Veterinary Medicine and Animal Science UNESP, Botucatu, São Paulo Department of Animal Reproduction and Veterinary Radiology School of Veterinary Medicine and Animal Science UNESP, Botucatu, São Paulo

A criopreservação dos espermatozóides eqüinos gera alterações nas membranas semelhantes às que ocorrem durante o processo de capacitação (criocapacitação). Sabe-se que o plasma seminal possui fatores decapacitantes. Além disso, é citado na literatura a possibilidade melhorar a preservação dos espermatozóides com membrana plasmática íntegra e motilidade, após refrigeração ou criopreservação, quando adicionado plasma seminal de animais que apresentam boa resposta à congelação ao sêmen de animais que apresentam espermatozóides com baixa capacidade de congelação. Assim, o presente experimento verificou se a criopreservação gera modificações semelhantes à capacitação e se a adição ao sêmen pós-descongelação de plasma seminal autólogo ou adquirido de um animal com boa resposta à congelação (homólogo) diminuía estes efeitos. Para isso, foram realizadas 5 colheitas de sêmen de 4 garanhões. O sêmen in natura foi avaliado e então, processado e submetido à congelação. Após a descongelação o sêmen foi novamente avaliado e então dividido em três tratamentos: adicionado de diluidor de criopreservação sem crioprotetor (DIL), deste com 20 % (v/v) de plasma seminal homólogo (PH) ou com 20 % (v/v) de plasma seminal autólogo (PA). Em seguida, o sêmen foi incubado em banho-Maria (37°C) e submetido as avaliações nos três diferentes períodos de amostragem (0, 60 e 120 minutos de incubação). As avaliações do sêmen consistiram na análise computadorizada das características da motilidade (CASA) e nas análises por citometria de fluxo da reação acrossomal, do potencial de membrana mitocondrial, da estabilidade da membrana plasmática, da peroxidação lipídica e na detecção da fosforilação do aminoácido tirosina presente na superfície da célula espermática. As variáveis foram submetidas à análise de variância e ao teste LSD para comparação das médias, ao nível de 5 % de significância. A criopreservação não aumentou a população de células viáveis com membrana acrossômica reagida, nem a população de células viáveis com aumento na desordem da membrana plasmática (capacitadas). Entretanto, a criopreservação levou a uma redução na quantidade de células com potencial de membrana mitocondrial e aumentou a peroxidação das membranas, além de induzir o aumento na fosforilação de proteínas presentes na superfície da célula. A criopreservação ocasionou uma redução de todas as variáveis analisadas pelo CASA, incluindo a população identificada como hiperativada. A adição do plasma seminal, indiferentemente da origem, melhorou a integridade da membrana plasmática, reduziu a população com aumento na desordem da membrana plasmática (capacitados), além de reduzir a quantidade de proteínas fosforiladas na superfície do espermatozóide. Porém esta melhora só foi encontrada nas análises iniciais (tempo 0), com o tempo o plasma seminal mostrou não ser um meio adequado para se manter o sêmen armazenado levando a um prejuízo na maioria das variáveis estudadas. Baseado nos resultados, pode-se sugerir a adição de plasma seminal com o intuito de reduzir os efeitos da criocapacitação. Contudo, ao adicionar o plasma seminal ao sêmen, este deve ser depositado no trato reprodutivo da fêmea o mais breve possível. Cryopreservation of equine spermatozoa leads to capacitation-like changes (cryocapacitation). Seminal plasma contains decapacitating factors. Moreover, it has been known that addition of seminal plasma from stallions with good post-thaw semen quality to semen of stallions with low post-thaw semen quality preserve progressive motility and membrane integrity better. Therefore, this study was intended to find out whether the cryopreservation process induces capacitation-like changes and if addition of autologous seminal plasma or seminal plasma from stallion with good post-thaw semen quality (homologous) reduces cryocapacitation effects. For this, five ejaculates were obtained from each of four mature stallions. Samples of in natura semen were analyzed, and submited to cryopreservation. Post-thawed semen was analyzed again so divided in three treatments: only cryopreservation media without cryoprotectants, cryopreservation media without cryoprotectants added 20 % (v/v) of seminal plasma homologous, and cryopreservation media without cryoprotectants added 20 % (v/v) of seminal plasma autologous. All samples were maintained in water bath (37°C) for 120 minutes, while semen analyzes were assessed at time 0, 60 and 120 min. The assessment of each sample was determined by computer-assisted sperm analyses of motility (CASA) and flow cytometry analyzes of acrosome reaction, mitochondrial potential, increased membrane fluidity, lipid peroxidation and degree of protein tyrosine phosphorylation in sperm surface. The variables were submitted to analysis of variance and LSD test at 5% level of significance. Cryopreservation did not increase viable cells with acrosome reaction, neither viable cells with increased membrane fluidity (capacitated). However, cryopreservation led an decrease of cell with mitochondrial potential and an increased in lipid peroxidation, besides increase tyrosine phosphorylation of the equine sperm surface proteins. The cryopreservation caused a reduction of all variables assessed by CASA, including hyperactivated cells. Seminal plasma addition, regardless of origin, improved integrity of plasma membrane, reduced number of viable cells with increased membrane fluidity, besides decrease phosphorylation of sperm surface proteins. Nevertheless, the improved created only was found in the initial analysis (time 0), seminal plasma shown a harmful effect in maintenance of sperm during incubation. Incubation of sperm in seminal plasma damaged majority of variables assessed. Based on results, seminal plasma could be added to post-thawed semen to decrease the cryocapacitation effects. However, post-thawed semen has to be deposited as soon as possible in female reproductive tract when the seminal plasma is added.