Summary: The aim of this work is to highlight the need of monitoring small populations to conserve their genetic variability by using a set of parameters to characterize both the structure of populations and management practices. As a representative example we analyse the pedigree information of the endangered Xalda sheep breed of Asturias. The herdbook of Xalda sheep included a total of 805 animals and 62 herds. The number of founders was 329. Nowadays, there are 562 live animals and 26 active herds. The breed is in risk of losing genetic diversity because of the abusive use of certain individuals as parents. The effective number of founder animals is 81.1. The effective number of founder herds is 9.9. The average value of inbreeding in the whole Xalda population was 1.5%. The average relatedness (AR) coefficient reached 1.8% in the whole pedigree. The genetic representation of the lines of founders is unbalanced. Inbreeding trends and effective size do not provide realistic information concerning the risk of loss of diversity as a result of the shallowness of the genealogical information. We suggest the monitoring of the breed using AR to unbalance the genetic contributions of specific individuals, equalizing the genetic representation of the founders and lines in the population. In addition, AR can suggest the introduction of new, under‐represented animals in herds showing high average AR values relative to the population. Our results can be useful to improve the development of conservation initiatives involving open herdbooks to avoid the risk of loss of genetic diversity caused by incorrect management practices.

According to the World Health Organization, infertility affects up to 14% of couples under reproductive age, leading to an exponential rise in the use of assisted reproduction as a route for conceiving a baby. In the same way, thousands of embryos are produced in cattle and other farm animals annually, leading to increased numbers of individuals born. All reproductive manipulations entail deviations of natural phenotypes and genotypes, with in vitro embryo technologies perhaps showing the biggest effects, although these alterations are still emerging. Most of these indications have been provided by animal models, in particular the bovine species, due to its similarities to human early embryo development. Oocytes and embryos are highly sensitive to environmental stress in vivo and in vitro. Thus, during in vitro culture, a number of stressful conditions affect embryonic quality and viability, inducing subfertility and/or long-term consequences that may reach the offspring. A high proportion of the embryos produced in vitro are arrested at a species-specific stage of development during the first cell divisions. These arrested embryos do not show signs of programmed cell death during early cleavage stages. Instead, defective in vitro produced embryos would enter a permanent cell cycle arrest compatible with cellular senescence, in which they show active metabolism and high reactive oxygen species levels. Later in development, mainly during the morula and blastocyst stages, apoptosis would mediate the elimination of certain cells, accomplishing both a physiological role in to balancing cell proliferation and death, and a pathological role preventing the transmission of damaged cells with an altered genome. The latter would acquire relevant importance in in vitro produced embryos that are submitted to stressful environmental stimuli. In this article, we review the mechanisms mediating apoptosis and senescence during early embryo development, with a focus on in vitro produced bovine embryos. Additionally, we shed light on the protective role of senescence and apoptosis to ensure that unhealthy cells and early embryos do not progress in development, avoiding long-term detrimental effects.

The aim of this study was to examine the effects of co-culture with Vero cells during the in vitro maturation (IVM) and three culture media, B2+5% fetal calf serum (FCS) on Vero cells, synthetic oviduct fluid (SOF)+5% FCS, and SOF+20 gL(-1) bovine serum albumin (BSA), on the developmental competence of the embryos and their ability to survive vitrification/warming. We also tested the effect of morphological quality and the age of the embryo on its sensitivity to vitrification. The IVM system neither affects the embryo development up to Day 7 nor survival rates after vitrification. The culture of embryos in SOF+FCS and in Vero cells+B2 allowed obtaining more Day 6 and Day 7 blastocysts, and a higher % of Day 7 blastocysts vitrified than culture in SOF+BSA. Contrarily, on Day 8, more blastocysts were vitrified in SOF+BSA than in SOF+FCS. Blastocysts quality affected development after vitrification/warming, and Day 7 embryos showed higher survival rates than their Day 8 counterparts. Day 7 blastocysts produced in Vero cells or in SOF+BSA survived at higher rates than those produced in SOF+FCS at 24 and 48 h after warming. Embryo culture with BSA allows obtaining hatching rates after vitrification/warming higher than those obtained after co-culture with Vero cells in B2 and FCS. Moreover, this system provides hatching rates from Day 8 blastocysts comparable to those obtained on Day 7 in Vero cells. Further studies, including embryo transfer to recipients, are needed to clarify factors affecting the freezability of in vitro produced bovine embryos.

The nuclear stage at which oocytes are cryopreserved influences further development ability and cryopreservation affects ultrastructure of both cumulus cells and the oocyte. In this work, we analyze the effects of vitrification at different nuclear and cytoplasmic maturation stages on the oocyte ultrastructure and developmental ability. Culture in TCM199 + PVA with roscovitine 25 M during 24h led to meiotic arrest (MA) in cumulus-oocyte complexes (COCs), while permissive in vitro maturation (IVM) was performed in TCM199, 10% FCS, FSH-LH and 17beta-estradiol for 24 h. Oocytes were vitrified using the open pulled straw method (OPS) with minor modifications. Fresh and vitrified/warmed COCs were fixed as immature, after IVM, after meiotic arrest (MA) and after MA + IVM. Vitrification combined with MA followed by IVM produced the highest rates of degeneration, regardless of the vitrification time. As a consequence, lower proportions of embryos cleaved in these groups, although differences were eliminated at the five-eight cell stage. Development rates up to day 8 were similar in all experimental groups, being significantly lower than those in fresh controls. Only oocytes vitrified after IVM were able to give blastociysts. The morphological alterations observed can be responsible for compromised development. More research is needed to explain the low survival rates of the bovine oocyte after vitrification and warming.

Parthenotes may represent an alternate ethical source of stem cells, once biological differences between parthenotes and embryos can be understood. In this study, we analyzed development, trophectoderm (TE) differentiation, apoptosis/necrosis, and ploidy in parthenotes and in vitro produced bovine embryos. Subsequently, using real-time PCR, we analyzed the expression of genes expected to underlie the observed differences at the blastocyst stage. In vitro matured oocytes were either fertilized or activated with ionomycin +6-DMAP and cultured in simple medium. Parthenotes showed enhanced blastocyst development and diploidy and reduced TE cell counts. Apoptotic and necrotic indexes did not vary, but parthenotes evidenced a higher relative proportion of apoptotic cells between inner cell mass and TE. The pluripotence-related POU5F1 and the methylation DNMT3A genes were downregulated in parthenotes. Among pregnancy recognition genes, TP-1 was upregulated in parthenotes, while PGRMC1 and PLAC8 did not change. Expression of p66shc and BAX/BCL2 ratio were higher, and p53 lower, in parthenotes. Among metabolism genes, SLC2A1 was downregulated, while AKR1B1, PTGS2, H6PD, and TXN were upregulated in parthenotes, and SLC2A5 did not differ. Among genes involved in compaction/blastulation, GJA1 was downregulated in parthenotes, but no differences were detected within ATP1A1 and CDH1. Within parthenotes, the expression levels of SLC2A1, TP-1, and H6PD, and possibly AKR1B1, resemble patterns described in female embryos. The pro-apoptotic profile is more pronounced in parthenotes than in embryos, which may differ in their way to channel apoptotic stimuli, through p66shc and p53 respectively, and in their mechanisms to control pluripotency and de novo methylation.