Lilian Rigatto Martins

Ultrastructural Characteristics of Non-matured and In Vitro Matured Oocytes Collected from Pre-pubertal and Adult Domestic Cat Ovaries

The present study describes the ultrastructural characteristics of cat oocytes before maturation and after 12- and 24-h in vitro maturation (IVM). Oocytes were recovered from pre-pubertal and adult queen ovaries after ovariohysterectomy and a proportion were stored in glutaraldehyde at 4 degrees C until examination by transmission electronic microscopy (TEM). Those selected for maturation were cultured before TEM in DMEM for 12 and 24 h at 38 degrees C in a humidified environment of 5% O(2), 5% CO(2) and 90% N(2). Specimens were divided into six groups: non-matured oocytes from pre-pubertal queens (PP0), non-matured oocytes from adult queens (A0), 12-h in vitro matured oocytes from pre-pubertal queens (PP12), 12-h in vitro matured oocytes from adult queens (A12), 24-h in vitro matured oocytes from pre-pubertal queens (PP24) and 24-h in vitro matured oocytes from adult queens (A24). Across the treatment groups, it was possible to observe differences in the thickness of the perivitelline space, the penetration of cumulus cell projections forming a junctional complex, distribution and density of small vesicles, lipid droplets, microvilli, mitochondria and cortical granules and variable degrees of development of Golgi complexes. These findings demonstrated that ultrastructural analysis of oocytes matured in vitro is a valuable tool to evaluate oocyte cytoplasmic maturation and that this IVM protocol was efficient in inducing gradual morphological changes necessary for cytoplasmic maturation of pre-pubertal and adult cat oocytes.

The seasonal and ovarian status effects on in vitro production of domestic cat embryos between Equator and Tropic of Capricorn

ABSTRACT.- Martins L.R., Fernandes , C.B., Villaverde A.I.S.B., Landim-Alvarenga F.C. & Lo-pes M.D.2014. The seasonal and ovarian status effects on in vitro production of do-mestic cat embryos in a region between Equator and Tropic of Capricorn. Pesquisa Veterinaria Brasileira 34(3):277-280 . Laboratorio de Reproducao Animal, Curso de Medici-na Veterinaria, Universidade Federal de Mato Grosso, Av. Alexandre Ferronato 1200, Sinop, MT 78557-267, Brazil. E-mail: lirigatto@yahoo.com.brFrom the Tropic of Capricorn to Equator, the seasonality of domestic cat is known to be absent, i.e., these animals are considered non-seasonal breeders at these regions. We hypothesized that this particularity might have some influence on in vitro embryo produc-tion. The aim of this experiment was to determine the percentage of cleavage and morulae and blastocyst formation produced from oocytes recovered from queen ovaries of three distinct status - follicular, luteal or inactive - during two different reproductive seasons experienced by cats in southeast of Brazil (22°53’09” S and 48°26’42” W) – non breeding season (NBS), comprehending January to March; and breeding season (BS), August to Oc-tober. Thirty queens were neutered. Ovaries were classified according to their status and were sliced in PBS for cumulus oocyte complex (COC) releasing. Grade I COC were wa-shed three times in H-MEM supplemented with BSA, glutamine, sodium pyruvate, cysteine, streptomycin and penicillin. Oocytes were incubated in groups of 20-30 in 400µL of DMEM supplemented with FSH, LH, estradiol, IGF-I and basic fibroblast growth factor under mine-ral oil for 30 or 36 hours at 38°C in humidified environment of 5% de O2, 5% CO2 and 90% N2. COC were fertilized in Ham’s F-10 medium supplemented with BSA, cysteine, pyruvate and streptomycin/penicillin (culture medium) with fresh semen selected through swim up technique. Eighteen hours later, the presumptive zygotes were denuded, the percentage of cleavage was determined and every 10 zygotes were transferred to 100 mL drops of culture medium for culture during three days. After 72 hours of culture the percentage of moru-lae formation was evaluated and these structures were transferred to drops of the same culture medium. At the eighth day of culture blastocyst formation was analyzed. During NBS, from a total of 272 (inactive), 162 (luteal) and 134 (follicular) fertilized oocytes, the percentage of cleaved zygotes, morulae and blastocysts derived from inactive ovaries were 24.63, 16.54 and 8.09 respectively; for those derived from luteal ovaries, the percentage was 21.6, 12.96 and 8.64, and for those from follicular ovaries, they were 24.62, 16.41 and 8.21. Considering BS, from a total of 102 (inactive), 198 (luteal) and 86 (follicular) fertilized oocytes, the relative frequency (%) of cleaved zygotes, morulae and blastocysts derived from inactive ovaries were 64.7, 41.17 and 23.53 respectively; for those derived from luteal ovaries, the percentage was 64.14, 40.41 and 23.73, and for those from follicular ovaries,

Influence of low-stress handling on reactivity score and pregnancy rate during fixed-time artificial insemination in Nellore cows

The objective of this work was to evaluate the influence of low-stress handling (LSH) on reactivity score and pregnancy rate during fixed-time artificial insemination (FTAI) for extensively raised Nellore cows. Multiparous cows (n = 126) were randomly allocated into two groups: G1 (n = 66) and G2, the control group (n = 60). Group G1 was subjected to LSH, in which the animals were handled in a calm and quiet manner, without loud noises or physical aggression, using the point of balance, respecting the flight zone and using flags to supplement handling. Group G2 was handled following the typical procedure, with yelling, kicking and the use of electric prods and sticks. On D0, D8 and D10, FTAI was performed in both groups. Reactivity was scored on D0, D8 and D10 at the squeeze chute, based on the tension score, breathing score, and bellowing score. Using the three criteria above, the reactivity scores were defined as follows: R1 (calm); R2 (slightly reactive); R3 (moderately reactive); R4 (reactive); and R5 (highly reactive). Thirty-five days after artificial insemination, pregnancy was determined using ultrasonography. There was no significant correlation between reactivity score and pregnancy rate in each group or between the pregnancy rates in both groups. However, there was a statistically significant difference between the G1 and G2 groups with regard to reactivity score (1.62±0.05 vs. 2.12±0.07). Low-stress handling influenced reactivity but did not affect pregnancy rate in extensively raised Nellore cows subjected to FTAI.

Ocorrência de hematomas em carcaças de bovinos transportados por duas distâncias

O objetivo foi determinar a ocorrencia de hematomas em carcacas de bovinos transportados por duas distâncias e abatidos no norte do estado do Mato Grosso. Foram avaliados 624 bovinos machos de 30 a 36 meses oriundos de distâncias variadas ate o frigorifico, sendo 352 animais oriundos de distâncias menores que 200km e 272 animais transportados por mais de 200km. Para avaliacao dos hematomas nas carcacas, seguiu-se o criterio descrito pela AUS-MEAT (2005), sendo as lesoes classificadas de acordo com a sua localizacao na carcaca (traseiro, dianteiro ou costado). A relacao de hematomas por grupo aumentou com a maior distância de transporte, sendo 43,75% e 95.58% de animais com hematomas para distâncias menores e maiores que 200 km, respectivamente. Quando analisada a regiao do hematoma, verificou-se 7,6 e 17,6 observacoes na regiao do dianteiro (P= 0,035) e 30,6 e 101,0 observacoes na regiao do traseiro (P=0,009) para distâncias menores e maiores que 200km, respectivamente. Portanto, a distância do transporte de bovinos ate o abate teve influencia sobre a quantidade de hematomas apresentados, principalmente na regiao do traseiro.

24 EVALUATION OF THE PREGNANCY RATE OF FIXED-TIME ARTIFICIALLY INSEMINATED BOS INDICUS COWS USING HETEROSPERMIC AND MONOSPERMIC SEMEN

The aim of this study was to establish a comparison between heterospermic and monospermic semen on the pregnancy rate of fixed-time AI Bos indicus cows. The experiment was conducted in the state of Mato Grosso, Brazil, during December 2010 and February 2011. Multiparous (n = 173) and primiparous (n = 204) Nelore cows, 45 ± 15 days postpartum, were evaluated on body condition score (BCS; score 1 to 9), and those that presented BCS between 5 and 8 were selected. Two protocols of estrous cycle synchronization were performed (PeCG and Pshang). Synchronization of the estrous cycle was achieved with the aid of the following protocol: on Day 0, all animals were treated with a progesterone vaginal implant with 1.9 g of progesterone combined with an IM injection of 2 mg of estradiol benzoate. On Day 7, an IM injection of 12.5 mg of dinoprost was administered. On Day 9, implants were withdrawn and the animals received an IM injection of 0.5 mg of estradiol cypionate. On Day 9, part of the animals (n = 222) received an IM injection of 300 IU mg of eCG (PeCG) and the other part (n = 155) had their calves removed for 48 hours (Pshang). All animals were inseminated 46 to 52 h after the end of the protocol. Cows were allocated randomly in 4 groups according to the donor male sample in the semen straw and groups were designated as MA (monospermic straw from bull A; n = 110), MB (monospermic straw from bull B; n = 102), MC (monospermic straw from bull C; n = 108), and HT (heterospermic sample from bulls A, B, and C; n = 57). Commercially available semen straws from Aberdeen Angus donors were used (ABS Pecplan, Brazil). Pregnancy diagnosis was performed 35 days after AI using real-time ultrasonography. Results are summarized in Table 1. There was no statistical difference between MA, MB, MC, and HT in any group according to the chi-square test (P > 0.05). However, there was a remarkable difference in pregnancy rates among the 3 bulls tested considering both protocols, probably due to the variation in the ovulation moment. Table 1.Pregnancy rates of Nelore cows submitted to FTAI protocol inseminated with monospermic (bull A, B, or C) or heterospermic (HT) semen

267 ULTRASTRUCTURAL CHARACTERISTICS OF NON-MATURED AND IN VITRO MATURED OOCYTES COLLECTED FROM FOLLICULAR, LUTEAL, AND INACTIVE OVARIES OF DOMESTIC CAT (FELIS CATUS) DURING BREEDING AND NON-BREEDING SEASON

The aim of this experiment was to describe the ultrastructural characteristics of non-matured (NM) and in vitro matured (IVM) cumulus–oophorus complexes (COC) recovered from adult queens during the breeding season (BS; July, August, and September) and the non-breeding season (NBS; January, February, and March) in southeast Brazil. Transmission electronic microscopy was performed in NM COC immediately after harvest, and IVM COC were matured for 36 h before transmission electronic microscopy. All IVM COC during both seasons were at metaphase II stage. Specimens were divided into COC from inactive ovaries, follicular ovaries, and luteal ovaries recovered during BS and NBS. During NBS, NM follicular and inactive COC presented a narrow perivitelline space covered with microvilli, which were less evident in NM luteal COC. Cumulus-cell projections penetrated the zona pellucida forming gap junctions with the oolemma in all NM COC. In the cytoplasm of NM inactive COC, lipid droplets and vesicles were evenly distributed in the ooplasm except for in the cortical zone, where clusters of mitochondria were observed. Non-matured luteal COC were also characterised by peripheral mitochondrial clusters, but greater clusters could also be seen centrally in the cytoplasm. In contrast, NM follicular COC were characterised by evenly distributed mitochondria within the ooplasma. In NM inactive and follicular COC, cortical granules were seen only in the central region of the cytoplasm, but the electron density of these organelles appeared to be low and Golgi complexes were often seen in association with these granules. The density of cortical granules in NM follicular COC was higher, but they were also present in central regions of the ooplasm. In all NM COC, a well-developed Golgi complex was observed. In all IVM COC, mitochondria clusters were no longer observed, and these organelles presented an even distribution toward the ooplasm. High-density cortical granules were present in the peripheral region of all IVM COC, although a small number could still be observed in central region of the ooplasm of IVM follicular COC. The perivitelline space was more prominent in IVM COC. During BS, COC characteristics were similar to NM COC during NBS; the presence of mitochondrial clusters associated with lipid droplets, well-developed cortical granules, and the presence of nuage could be observed, except that all cortical granules located in the central region of the cytoplasm of NM follicular COC during BS. A great amount of smooth endoplasmic reticulum could be verified in NM follicular and luteal COC. During BS, all IVM COC presented uniform distribution of mitochondria and peripheral distribution of cortical granules in the ooplasma. These results indicate that IVM was efficient in inducing the morphological changes necessary for cytoplasmic maturation of cat COC, independently of the ovarian status. The authors acknowledge FAPESP.

Effect of plasma progesterone concentration on recovery and in vitro maturation of oocytes obtained from buffalo ovaries: partial results

The spermatic changes associated with the natural infection in dogs by Leishmania sp was evaluated during eight consecutive weeks, using ejaculates of six seronegative and six seropositive dogs. The samples were collected once a week and evaluated for volume, concentration, motility, vigor, sperm morphology, chromatin integrity, simultaneous evaluation of the plasmatic membrane integrity, acrosome, and mitochondrial potential. The total proteins of the seminal plasma and blood were measured. The visceral leishmaniasis caused increase of major and minor defects in spermatozoa of animals attacked by moderate to severe stages of the disease. In more advanced stages of the illness, the acrosomal and plasmatic membranes integrity was adversely affected. It was not possible to establish a pattern refering the evaluation of the mitochondrial potential. The incidence of morphological changes in the seropositive animals did not promote an increase of injuries to the chromatin. All animals with leishmaniasis presented hyperproteinemia of the semen.