Bovid Interspecies Somatic Cell Nuclear Transfer with Ooplasm Transfer.

Interspecies somatic cell nuclear transfer (iSCNT) contributes to the preservation of endangered species, albeit nuclear-mitochondrial incompatibilities constrain its application. iSCNT, coupled with ooplasm transfer (iSCNT-OT), has the potential to overcome the challenges associated with species- and genus-specific differences in nuclear-mitochondrial communication. Our iSCNT-OT protocol combines the transfer of both bison (Bison bison bison) somatic cell and oocyte ooplasm by a two-step electrofusion into bovine (Bos taurus) enucleated oocytes. The procedures described herein could be used in further studies to determine the effects of crosstalk between nuclear and ooplasmic components in embryos carrying genomes from different species.

Impact of negative energy balance and postpartum diseases during the transition period on oocyte quality and embryonic development in dairy cows.

Transition period is a critical time for dairy cows because a large proportion of clinical and subclinical diseases are observed in the first month after parturition. Occurrence of negative energy balance is associated with depressed immunity and these conditions can affect oocyte quality and further embryonic development. The aim of this study was to assess the effects of negative energy balance-associated disorders on in vitro embryo production (IVP) in dairy cattle. We hypothesized that subclinical metabolic and/or inflammatory disorders have a negative effect on oocyte developmental competence and morphokinetic parameters of the resulting embryos. The study was conducted on 30 lactating Holstein-Friesian cows which were assigned into four groups: healthy (HEAL, n = 6), metabolic disease (META, n = 8), inflammatory disease (INFL, n = 8), or combined metabolic and inflammatory disease (COMB, n = 8). Ovum pick-up (OPU) was performed twice weekly on all cows over a period of four weeks (n = 8 OPU sessions/cow) starting on the fifth week postpartum, and the collected oocytes were subjected to routine IVP. Donor's health status did not affect the number of oocytes/OPU or the recovery rate (p > 0.05). The number of quality 1 oocytes collected from INFL and COMB cows was lower compared to HEAL cows (p < 0.05). Also, the percentage of quality 1 embryos was reduced in META and COMB compared to HEAL cows (p < 0.05). Cleavage, blastocyst and hatching rates were similar among groups (p > 0.05). Presence of disease did not affect the time required by zygotes to reach specific developmental stages, as recorded by means of time-lapse monitoring. Nevertheless, there was a higher probability of direct cleavage after IVF in oocytes of COMB cows compared to those of HEAL cows (p < 0.05). In conclusion, oocytes and embryos derived from dairy cows diagnosed with subclinical metabolic and/or inflammatory diseases during the transition period showed reduced quality but similar developmental potential and morphokinetics when compared to healthy cows. These results shed light on the consequences of subclinical disease on embryonic development in dairy cows which might be important for embryo transfer programs.

Evaluation of different acute-phase proteins for herd health diagnostics in early postpartum Holstein Friesian dairy cows.

This research communication describes (1) the comparison of acute-phase protein (APP) concentrations in transition dairy cows on different farms using both pooled and individual blood samples, and (2) the association among different APP and clinical health parameters. The first hypothesis was that early postpartum dairy cows from different farms differ in the level of inflammation, which might be determined using APP assays in both pooled and individual blood samples. The second hypothesis was that the APP haptoglobin (Hp) might be the most sensitive parameter to detect cows at risk of excessive postpartum inflammation concomitant with systemic disease. Serum concentrations of Hp, serum amyloid-A (SAA), total protein (TP), albumin (Alb), coeruloplasmin (Cp) and C-reactive protein (CRP) in 100 fresh lactating cows (within 0-8 d postpartum) from 10 farms were compared and associated to clinical health parameters (rectal body temperature, vaginal discharge (Metricheck™ score), rumen fill, vulvovaginal laceration) using both pooled and individual blood samples. Mean serum concentrations of Hp, SAA and TP revealed significant differences among farms. Pooled serum samples of farms showed high correlations with the mean of individual samples. Only Hp was significantly positively correlated to both body temperature and Metricheck™ score. In conclusion, Hp differentiates dairy farms regarding the inflammatory state of transition cows using individual and pooled serum samples within the first week postpartum. It also mirrors the individual degree of inflammation, thus proving to be a diagnostic parameter of high interest during the periparturient period.

Short communication: Repeatability of intravenous glucose tolerance test traits in young Holstein-Friesian cattle.

Few studies have compared individual variability of an intravenous glucose tolerance test (ivGTT) obtained daily in young cattle. The objective of this study was to evaluate the repeatability of glucose traits and insulin responses derived from ivGTT during 3 consecutive days in Holstein-Friesian cattle (bulls: n = 4, steers: n = 3, heifers: n = 3). Blood collections were performed from min 0 (basal concentrations) to min 63 (last measurement). Additional estimates included maximal concentration for glucose and insulin, glucose half-life time (GHLT), and glucose and insulin area under the curve. Glucose traits showed higher repeatability when compared with insulin responses. On average, the glucose trait and insulin response with the lowest coefficient of variation was GHLT (0.7%) and insulin area under the curve (21.3%), respectively. In addition, a general linear model with repeated measures was used to test for significant differences in glucose and insulin concentrations over time at different trial days. Both glucose and insulin concentrations were influenced by time point but not by trial day or time point × trial day. The results from this study showed that individual insulin responses were more variable than glucose traits. The high repeatability of GHLT evidenced the strict regulatory glucose disposal mechanisms occurring in young Holstein-Friesian cattle, whereas insulin responses showed high variability despite controlled management and nutritional practices.

Evaluation of glucose dose on intravenous glucose tolerance test traits in Holstein-Friesian heifers.

Glucose metabolism in dairy and beef cattle has received considerable attention because balanced blood glucose is essential for numerous processes, such as milk production and general health. The glucose tolerance test measures the ability of an organism to regulate blood glucose levels. Glucose half-life time (GHLT) has high heritability and could serve as a potential parameter to breed for metabolic resistance. However, studies focusing on identification of an adequate glucose dose have not yet been conducted in cattle. The objective of this study was to analyze the effect of 5 different glucose doses (0.5, 1, 1.5, 2, and 3 g/kg of body weight0.75) on intravenous glucose tolerance test (ivGTT) traits and insulin responses in nongestating heifers. A total of 150 tests were performed in 30 Holstein-Friesian heifers aged 13 to 15 mo. Blood samples were obtained every 7 min after glucose injection until min 63. Glucose traits and insulin parameters included blood serum glucose and insulin concentration at min 0 (basal concentration), min 7 to 21 (peak glucose and insulin concentration), and min 63 (last sampling) relative to glucose administration, glucose and insulin area under the curve (GAUC and IAUC), and GHLT estimated between min 14 and 42. Serum glucose and insulin concentrations were measured according to the hexokinase colorimetric method and radioimmunoassay, respectively. Generalized linear mixed model was used to test for significant differences in ivGTT traits, insulin responses, and glucose elimination rates (k) over time at different glucose doses. Maximum glucose and insulin concentrations at min 63 increased with higher glucose doses. Significantly lower GHLT were obtained at increasing glucose doses, whereas GAUC and IAUC were significantly higher at increasing doses. The k values were affected by glucose dose and by time interval. Glucose dose greatly affected most ivGTT traits, insulin responses, and glucose elimination rates. Therefore, researchers should standardize their methods to achieve repeatable results and use the same time points for GHLT calculation. Higher glucose doses (≥1.5 g/kg of body weight0.75) triggered glucose concentrations above the glucose renal threshold during the initial 42 min, whereas the lowest glucose concentration failed to induce a maximum insulin response. Further research is necessary to determine an adequate dose inducing maximum insulin responses with minimum renal glucose losses.

Influence of food deprivation on intravenous glucose tolerance test traits in Holstein Friesian heifers.

Glucose is essential for numerous cellular functions due to its involvement in energy supply from early development to adulthood. In the lactating cow, glucose demands by the mammary gland significantly increase to support milk production when compared with other tissues. Thus, insufficient blood glucose levels might predispose dairy cows to metabolic disturbances. The intravenous glucose tolerance test (ivGTT) is a suitable tool to characterize glucose metabolism and insulin responses, but results must be reliable and repeatable. One factor influencing ivGTT is food deprivation period, which has been considered as an obligatory requirement before conduction of glucose tolerance studies in monogastric species, whereas it has been permissive in ruminants. The objective of this study was to determine the influence of 5 fasting periods (0, 12, 24, 36, and 48 h) on ivGTT traits and insulin responses in German Holstein heifers. A total of 140 tests were conducted in 28 females aged 12 to 19 mo. Blood samples were collected every 7 min within 1 h. Assessed glucose and insulin parameters included basal serum glucose and insulin concentration, maximum glucose and insulin concentration obtained between min 7 to 21, and concentrations at min 63 (last sampling) relative to glucose administration, glucose area equivalent (GA), glucose area under the curve (GAUC), insulin area equivalent (InsA), insulin area under the curve (InsAUC), and blood glucose half-life time (GHLT). Serum glucose and insulin concentration were measured according to the hexokinase colorimetric method and solid phase radio immunoassay, respectively. The generalized linear mixed model was used to test for significant differences in all glucose traits and insulin responses at different fasting periods. The model used season and weight as confounding variables. Glucose and insulin concentrations at 0, 7 to 21 (maximum concentration), and 63 min were affected by the duration of food deprivation. The GA, InsA, GAUC, InsAUC, and especially GHLT were also affected by fasting period. A positive linear relationship between GHLT and length of food deprivation was found. Significantly higher GA, GAUC, GHLT values, and glucose and insulin concentration at min 63 were obtained at increasing fasting periods. High intraclass correlation coefficient (0.48) was found for GHLT. The results demonstrate that GHLT might be used to reliably characterize an individual's glucose metabolism and the importance of standardizing food deprivation schedules when performing an ivGTT in nongestating Holstein heifers.

Lack of effects of ooplasm transfer on early development of interspecies somatic cell nuclear transfer bison embryos.

BACKGROUND: Successful development of iSCNT (interspecies somatic cell nuclear transfer) embryos depends on complex interactions between ooplasmic and nuclear components, which can be compromised by genetic divergence. Transfer of ooplasm matching the genetic background of the somatic cell in iSCNT embryos is a valuable tool to study the degree of incompatibilities between nuclear and ooplasmic components. This study investigated the effects of ooplasm transfer (OT) on cattle (Bos taurus) and plains bison (Bison bison bison) embryos produced by iSCNT and supplemented with or without ooplasm from cattle or plains bison oocytes. RESULTS: Embryos in all groups were analysed for developmental competence that included cleavage rates, ATP content, and expression of nuclear- and mitochondrial- encoded genes at 8-16 cell stage. Interestingly, no significant differences were observed in embryo development, ATP content, and expression of nuclear respiratory factor 2 (NRF2), mitochondrial transcription factor A (TFAM) and mitochondrial subunit 2 of cytochrome c oxidase (mt-COX2) among groups. Thus, although OT did not result in any detrimental effects on the reconstructed embryos due to invasive manipulation, significant benefits of OT were not observed up to the 8-16 cell stage. CONCLUSIONS: This study showed that a viable technique for OT + SCNT is possible, however, further understanding of the effects of OT on blastocyst development is necessary.

Developmental competence of 8?16-cell stage bison embryos produced by interspecies somatic cell nuclear transfer.

Altered communication between nuclear and cytoplasmic components has been linked to impaired development in interspecies somatic cell nuclear transfer (iSCNT) embryos as a result of genetic divergence between the two species. This study investigated the developmental potential and mitochondrial function of cattle (Bos taurus), plains bison (Bison bison bison) and wood bison (Bison bison athabascae) embryos produced by iSCNT using domestic cattle oocytes as cytoplasts. Embryos in all groups were analysed for development, accumulation of ATP, apoptosis and gene expression of nuclear- and mitochondrial-encoded genes at the 8-16-cell stage. The results of this study showed no significant differences in the proportion of developed embryos at the 2-, 4- and 8-16-cell stages between groups. However, significantly higher ATP levels were observed in cattle SCNT embryos compared with bison iSCNT embryos. Significantly more condensed and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL)-positive nuclei were found in plains bison iSCNT embryos. No significant differences in the expression levels of nuclear respiratory factor 2 (NRF2) or mitochondrial subunit 2 of cytochrome c oxidase (mt-COX2) were found in any of the groups. However, mitochondrial transcription factor A (TFAM) expression significantly differed between groups. The results of this study provide insights into the potential causes that might lead to embryonic arrest in bison iSCNT embryos, including mitochondrial dysfunction, increased apoptosis and abnormal gene expression.

Somatic cells, stem cells, and induced pluripotent stem cells: how do they now contribute to conservation?

More than a decade has now passed since the birth of the first endangered species produced from an adult somatic cell reprogrammed by somatic cell nuclear transfer. At that time, advances made in domestic and laboratory animal species provided the necessary foundation for attempting cutting-edge technologies on threatened and endangered species. In addition to nuclear transfer, spermatogonial stem cell transplantation and induction of pluripotent stem cells have also been explored. Although many basic scientific questions have been answered and more than 30 wild species have been investigated, very few successes have been reported. The majority of studies document numerous obstacles that still need to be overcome to produce viable gametes or embryos for healthy offspring production. This chapter provides an overview of somatic cell and stem cell technologies in different taxa (mammals, fishes, birds, reptiles and amphibians) and evaluates the potential and impact of these approaches for animal species conservation.