José Ernesto Hernández Pichardo

Semen is a complex fluid that, in addition to spermatozoa, contains other cell populations, including immune cells, immature male germ cells, epithelial cells, and fibroblasts. These cells share the diploid condition, making them suitable candidates as nuclear donors for somatic cell nuclear transfer (SCNT) cloning. The generation of viable embryos and offspring has been demonstrated using these cells. Effective methods for isolating them from semen include centrifugation and osmotic gradient techniques; however, prolonged in vitro culture periods are necessary to establish primary cultures from these isolated cells. Furthermore, the samples that were obtained contained a high load of spermatozoa, which interferes with the establishment and maintenance of in vitro cultures. To date, primary cultures have only been successfully established from fresh semen samples, while attempts using cryopreserved semen have consistently failed. This limitation significantly restricts the potential to generate clones from cryopreserved semen samples. The present study proposes a simple microaspiration-based methodology for isolating somatic cells from cryopreserved equine semen. This approach allows for the rapid retrieval of a sufficient number of cells for immediate use in SCNT cloning procedures.

Semen is a complex fluid that, in addition to spermatozoa, contains other cell populations, including immune cells, immature male germ cells, epithelial cells, and fibroblasts. These cells share the diploid condition, making them suitable candidates as nuclear donors for somatic cell nuclear transfer (SCNT) cloning. The generation of viable embryos and offspring has been demonstrated using these cells. Effective methods for isolating them from semen include centrifugation and osmotic gradient techniques; however, prolonged in vitro culture periods are necessary to establish primary cultures from these isolated cells. Furthermore, the samples that were obtained contained a high load of spermatozoa, which interferes with the establishment and maintenance of in vitro cultures. To date, primary cultures have only been successfully established from fresh semen samples, while attempts using cryopreserved semen have consistently failed. This limitation significantly restricts the potential to generate clones from cryopreserved semen samples. The present study proposes a simple microaspiration-based methodology for isolating somatic cells from cryopreserved equine semen. This approach allows for the rapid retrieval of a sufficient number of cells for immediate use in SCNT cloning procedures.

Somatic cell nuclear transfer (SCNT) has great potential for the replication of high-commercial-value animals, threatened wild species for conservation purposes, and transgenic animals for biomedical purposes. However, SCNT has a low success rate due to intrinsic factors of the technique itself, which leads to low rates of embryonic development and epigenetic alterations in cloned embryos. The objective of this study was to evaluate the effect of OF–UF on the intracellular concentrations of ROS and GSH and the development of cloned and parthenogenetic Ovis aries embryos. The results do not show a beneficial effect on the development of parthenogenetic and cloned embryos at concentrations of 0.5% OF–UF. Furthermore, at 1% OF–UF, an adverse effect was observed in cloned embryos at the blastocyst stage and 2% OF and UF in parthenogenetic embryos during the first divisions. Decreases in ROS and GSH levels were observed in the parthenogenetic blastocysts treated with 1% OF–UF, but not in the clones, in which a higher concentration of GSH and a similar concentration of ROS were observed. No effect of OF–UF was observed on embryonic development and redox balance in sheep embryos cloned via handmade cloning.

Somatic cell nuclear transfer (SCNT) has great potential in the replication of high commercial value animals, threatened wild species for conservation purposes, and transgenic animals for biomedical purposes. However, SCNT has a low success rate, due to intrinsic factors of the technique itself, which will lead to low rates of embryonic development and epigenetic alterations in the cloned embryos. The objective of the present study was to evaluate the effect of OF-UF on the intracellular concentration of ROS, GSH, and the development of Ovis aries cloned and parthenogenetic embryos. The results did not show a beneficial effect on the development of parthenogenetic and cloned embryos at concentrations of 0.5% OF-UF. Furthermore, at 1% OF-UF, an adverse effect was observed in cloned embryos at the blastocyst stage and 2% OF and UF in parthenogenetic embryos in the first divisions. A decrease in ROS and GSH levels was observed in the parthenogenetic blastocysts treated with 1% OF-UF, but not in the clones, in which a higher concentration of GSH and a similar concentration of ROS was observed. No effect was observed of OF-UF on embryonic development and redox balance on sheep clone embryos by Handmade Cloning.

Vaginal cytology is a complementary economic method and of simple realization, an indicative to determine in which stage of the estrous cycle the dog is, to achieve a higher fertility and fertility rate. This method is based on determining the type and quantity of cells of the different stages of the estrous cycle, since the hormonal changes that the vaginal mucosa undergoes during the estrous cycle are shown in the morphology of its epithelial cells. The canine female in her reproductive life goes through different phases of activity and hormonal rest that are repeated cyclically. This is called the estrous cycle and consists of 4 stages: proestrus, estrus, diestrus and anestrus. The interpretation of vaginal cytology’s, is a process to which a considerable amount of time is dedicated by its observation in the microscope and the same interpretation by the doctor which can become subjective and poorly performed, causing economic losses for the owners. Therefore, this work proposes an automatic system that will identify six types of cells and the quantity of them in the glass slide, based on a Faster R-CNN to determine in which stage of the estrous cycle the dog is. Our results show an accuracy of 91.6%. The proposed system will improve the efficiency and speed of cytology’s to decreased from 1 h approximately to just a few seconds.