Ana Izabel Silva Balbin Villaverde

Comparison of efficiency between two artificial insemination methods using frozen-thawed semen in domestic cat (Felis catus): artificial insemination in domestic cats.

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.

Cryoprotective effect of different glycerol concentrations on domestic cat spermatozoa

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.

Blood and seminal plasma concentrations of selenium, zinc and testosterone and their relationship to sperm quality and testicular biometry in domestic cats

The aim of this study was to assess seminal plasma (SP) and serum concentrations of zinc (Zn), selenium (Se) and testosterone (T) in domestic cats and determine whether these are related to sperm quality and testicular biometry. Six tomcats were collected using an artificial vagina and sperm analysis included motility by CASA, morphology, plasma membrane integrity, and sperm count. Serum and SP were submitted to total T concentration determination using a solid-phase radioimmunoassay technique while Zn and Se were measured by atomic absorption spectroscopy. Serum T concentrations were greater compared to SP concentrations, but both values were significantly correlated. Se concentrations were higher in serum, whereas SP had greater Zn values. Concentrations of Se, Zn and T were not correlated with each other either in serum or SP. Negative correlations were detected between Se concentrations in SP and total sperm head defects, and between Se concentrations in serum and VAP, VSL, STR, and LIN. Serum concentrations of Zn were negatively correlated with total abnormal sperm and midpiece defects and positively related to progressive motility. Both serum and SP concentrations of T had no relationship with sperm quality. Concentrations of Se exhibited a negative correlation with total testicular weight, whereas T concentrations in SP and serum were correlated with total testicular volume and weight. In conclusion, both Se and Zn concentrations in serum were correlated to sperm quality variables in the domestic cat, thus, making these potential candidates for fertility markers.

High incidence of 'Dag-like' sperm defect in the domestic cat.

The occurrence of a high incidence of sperm tail defects in a male domestic cat resembling the known ‘Dag-like’ defect is reported. Sperm analyses were performed in ejaculated samples collected by an artificial vagina and in testicular and epididymal sperm cells after castration. The following alterations were observed using transmission electron microscope: heavily coiled sperm tails containing several axonemal units enclosed in the same common cell membrane; aberrations in the axonemal main structure; and swollen and unevenly distributed mitochondria in the midpiece. Abnormal modifications in the mitochondrial sheath were also found in sperm cells retrieved from testes and epididymides. Considering these findings, we can conclude that this is the Dag-like defect, described previously in other domestic species and a testicular origin may be involved.

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,

Correlations of zinc and selenium concentrations in seminal plasma and serum with sperm count and morphology in domestic cats

Univ Estadual Paulista, Dept Clin Surg & Anim Reprod, Fac Vet Med, UNESP, Sao Paulo, BrazilUNESP, Sch Vet Med & Anim Sci, Dept Anim Reprod, Lab Adv Reprod & Cell Therapy LAN A, Botucatu, SP, Brazil

Effect of different glycerol concentrations on the viability of domestic cat frozen-thawed semen

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.