Xanthan as an additive external cryoprotectant for frozen ram semen

The xanthan gum can contribute to reduce the formation of ice crystals in the sperm freezing solution, facilitating the preservation of sperm viability after thawing. However, there is no information about the use of xanthan gum as an additive for cryoprotection of ram sperm. This study aimed to determine the effect of three different concentrations of xanthan (0.15%, 0.20% and 0.25%) on post-thawing ram sperm quality. In vitro tests, evaluating sperm motility and vigor, membrane and acrossome integrity and mitochondrial functionality were conducted before freezing and after thawing. Biochemical tests were performed to evaluate reactive oxygen species (ROS) and total antioxidant capacity (TOSC) by using a fluorometer and the fluorescent probe H2DCF-DA. The addition of xanthan did not benefit sperm membrane and acrossome integrity and mitochondrial functionality (p>0.05), but the post-thawing sperm motility was lower (p0.05), mas a motilidade espermática pós-descongelamento foi reduzida (p<0.05) nos tratamentos com 0,20 e 0,25% de xantana (22,2% ± 4,1 e 20,8% ± 4,1, respectivamente), em comparação com o controle sem inclusão de xantana (32,2% ± 4,1) (p<0.05). Por outro lado, a xantana mostrou características antioxidantes, pois a produção de EROs foi reduzida conforme aumentou a concentração da xantana (p<0.05). Assim, a xantana apresentou capacidade antioxidante extracelular, sendo eficiente na diminuição das espécies reativas de oxigênio geradas no processo de congelamento

Fertility preservation of ovarian germ cells: the horse and deer models

Preserving viability of frozen gametes and reproductive tissues is crucial to understand and overcome species-specificities in respect to the diversity in cryobiological properties and requirements among cell types and tissues. The use of different animal models to study ovarian tissue cryopreservation will help to uncover several important factors related to germ cells preservation. Horses (Equus ferus caballus) have been proven to be an excellent model for reproductive biology studies with implications for humans. White-tailed deer (Odocoileus virginianus) are one of the most abundant wild species in the United States, but little information about their reproductive features are known. Therefore, five studies were conducted in this Dissertation with the following general objectives: (i) to develop ovarian tissue cryopreservation techniques for horses and white-tailed deer species; and (ii) to determine the effects of ovarian tissue cryopreservation techniques on morphological and molecular mechanisms related to folliculogenesis in horse and white-tailed deer species. In study one, equine ovarian tissue was used to determine the ideal ovarian fragment size for better cooling resistance under storage at 4°C. In study two, equine ovarian tissues were used to determine the toxicity effect of cryoprotective agents on ovarian tissue pre- and post-cryopreservation. In study three, equine ovarian tissues were used to compare slow-freezing versus vitrification; and to determine the best cryoprotective agents for each cryopreservation method. In study four, white-tailed deer reproductive tracts were used to characterize the age effect on reproductive features. In study five, white-tailed deer ovarian tissue was used to compare slow-freezing versus vitrification methods to preserve preantral follicles under in vitro culture. The main findings of the horse studies were: (i) equine ovarian tissue can be stored at 4°C for up to 24 h when biopsy ovarian fragments are used; (ii) ethylene glycol seems to be a less harmful cryoprotectant agent to equine preantral follicles; and (iii) both slow-freezing and vitrification methods similarly preserved the follicle morphology after time of culture. The main findings of the white-tailed deer studies were: (i) aging caused quantitative and qualitative effects on the ovarian reserve of white-tailed deer; (ii) fresh ovarian tissue can be cultured for up to seven days preserving the tissue integrity; and (iii) fragments cryopreserved by vitrification had higher follicle viability during in vitro culture than by the slow-freezing method. In conclusion, this work demonstrated the viability to cryopreserve equine and white-tailed deer ovarian tissue. Furthermore, the frozen-thawed equine and white-tailed deer ovarian tissue can be cultured for up to seven days

Association of the occurrence of some diseases with reproductive performance and milk production of dairy herds in southern Brazil

This study analyzed associations between the occurrence of mastitis, metritis and retained placenta with the reproductive performance and milk production of four Holstein dairy herds of southern Brazil. Calving-to-conception intervals (CCI) and daily milk production were compared across herds, number of artificial inseminations and clinical occurrences and occurrence of mastitis, metritis and retained placenta. The CCI of cows with three or more clinical occurrences was longer than for those with no occurrences, but shorter for cows with retained placenta than for those without it. Cows with clinical mastitis up to 60 days post-partum had shorter CCI than those with mastitis after 60 days. Cows with retained placenta also had higher frequency of metritis. The daily milk production was higher for cows with three or more clinical occurrences than for those with one or none and for those without retained placenta than for those with retained placenta. A high number of clinical occurrences was related to prolonged CCI and increased daily milk production

Cryprotectant effect of trehalose and low-density lipoprotein in extenders for frozen ram sperm

This study tested trehalose and low-density lipoprotein (LDL) as cryoprotectants in extenders for frozen ram semen. In the first experiment, the extenders were Tris, with 20% egg yolk (E1-1); E1-1 with 5% glycerol (E1-2); E1-1 with 100mM trehalose (E1-3); and E1-1 with 100mM trehalose and 5% glycerol (E1-4). Sperm motility and membrane integrity of the E1-2, E1-3 and E1-4 extenders were greater than for E1-1 (P < 0.05), but acrosome integrity following cryopreservation did not differ. In the second experiment, the extenders were Tris, with 20% egg yolk and 100mMtrehalose (E2-1); Tris with 8% LDL and 5% glycerol(E2-2); Tris with 8% LDL and 100mM trehalose (E2-3); and Tris with 8% LDL, 100mM trehalose and 5% glycerol (E2-4). Sperm membrane integrity was lowest for the E2-1 extender (P < 0.05), but similar for extenders including LDL. Sperm motility post-thawing was highest for E2-2 and E2-3 extenders (P < 0.05), but acrosome integrity did not differ. Thus, extenders including trehalose and LDL as cryoprotectants recorded a post-thawing ram sperm quality similar to that achieved when using conventional cryoprotectants

Efeito do álcool polivinílico na criopreservação de espermatozóides de ovinos

O processo de criopreservação causa lesões nos espermatozóides de ovinos, por este motivo desenvolvimento de diluentes que incluam crioprotetores que minimizem estas lesões se justifica. Este trabalho objetivou avaliar à adição de álcool poliviliníco na composição de um diluente utilizado para congelamento do sêmen. Foram utilizados 6 carneiros SRD, totalizando 30 ejaculados. As amostras foram acondicionadas a 5°C com tris glucose e, somente após 1 hora, era acrescido 0,05% PVA. A motilidade espermática foi avaliada durante 30 minutos, em intervalos de 2,5 min entre cada análise. A comparação da motilidade em função do tempo foi realizada através da análise de variância de Kruskal-Wallis. A motilidade observada em 2,5 min após a adição do PVA foi de 70,5% , após este período a motilidade foi decrescente. O tempo máximo de exposição ao PVA, antes do congelamento, seria de 5 minutos onde a motilidade foi de 46,5%. Portanto, é possível concluir que o tempo de exposição da célula espermática a um diluente contendo PVA na concentração de 0,05% não pode exceder 5 min, preservando dessa forma a qualidade seminal antes da criopreservação