Whole blood transcriptome analysis reveals footprints of cattle adaptation to sub-arctic conditions.

Indigenous cattle breeds in northern Eurasia have adapted to harsh climate conditions. The local breeds are important genetic resources with cultural and historical heritages, and therefore, their preservation and genetic characterization are important. In this study, we profiled the whole-blood transcriptome of two native breeds (Northern Finncattle and Yakutian cattle) and one commercial breed (Holstein) using high-throughput RNA sequencing. More than 15 000 genes were identified, of which two, 89 and 162 genes were significantly upregulated exclusively in Northern Finncattle, Yakutian cattle and Holstein cattle respectively. The functional classification of these significantly differentially expressed genes identified several biological processes and pathways related to signalling mechanisms, cell differentiation and host-pathogen interactions that, in general, point towards immunity and disease resistance mechanisms. The gene expression pattern observed in Northern Finncattle was more similar to that of Yakutian cattle, despite sharing similar living conditions with the Holstein cattle included in our study. In conclusion, our study identified unique biological processes in these breeds that may have helped them to adapt and survive in northern and sub-arctic environments.

Production and manipulation of bovine embryos: techniques and terminology.

There are numerous publications regarding bovine embryos, ranging from descriptions of their appearance and development to emerging techniques in the field of assisted reproductive technology. Concurrently, several specialized terms have been developed to describe the bovine embryo. The purpose of the current review is two-fold; it is primarily to describe techniques involved in the in vivo and in vitro production of bovine embryos and their manipulation, and secondarily to summarize specialized terms used in these processes. The intention is not to review these techniques in detail, but instead to provide salient points and current knowledge regarding these techniques, with a focus on terminology. The first review dealt with classical and contemporary terminology used to describe morphologic aspects of ovarian dynamics in cattle. Subsequently, the terms and current understanding of processes involved in preattachment bovine embryos were described in the second review. As the third article in a series, this mini-review is focused on defining the production, manipulation, and transfer of bovine preattachment embryos.

Optimal utilization of modern reproductive technologies to maximize the gross margin of milk production.

In this study, a linear programming model was developed to maximize the gross margin of milk production by determining the optimal use of different reproductive technologies in a dairy herd. The model has the potential to vary the use of conventional artificial insemination, insemination with X-sorted sperm, and the use of unselected or sex-selected embryo recovery and transfer. Data from Finnish dairy herd recording systems were used to parameterize the model. This paper presents the results of 6 scenarios for a herd size of 60 dairy cows. In the basic scenario, the optimum economic combination for Finnish conditions was to inseminate 10 heifers and 22 cows with unsorted semen, 8 heifers with X-sorted sperm, and to use 20 cows as embryo donors which was the upper constraint for this technique. The embryo donors were inseminated with conventional semen for both embryo production and their subsequent pregnancy. Without restriction on embryo recovery, the optimum combination was to use all heifers as donors of sex-selected embryos and all cows as donors of unselected embryos. It was more profitable to produce female embryos with X-sorted sperm than by sorting embryos. Embryo recipients were not economically justified in any scenario. In practice, the optimal strategy is herd-specific depending on the input costs, output values and the technical success of each reproductive technology in that herd. This single-year linear programming model adequately differentiates between breeding technologies within a herd, but further research is needed to develop dynamic models to consider genetic improvement and herd expansion.

The effect of ascorbic acid during biopsy and cryopreservation on viability of bovine embryos produced in vivo.

Multiple ovulation embryo transfer (MOET) is used to make more rapid progress in animal breeding schemes. On dairy farms, where female calves are more desired, embryo sex diagnosis is often performed before embryo transfer. Fresh transfers have been favored after biopsy due to cumulative drop in pregnancy rates following cryopreservation. The aim of this study was to explore whether exposure to ascorbic acid (AC) during biopsy and freezing increases the viability of biopsied embryos after cryopreservation. Data on presumptive pregnancy and calving rates of biopsied and cryopreserved/overnight-cultured embryos were gathered. Results showed differences in presumptive pregnancy rates between the groups: 45% for both biopsied-cryopreserved groups (control and AC), 51% for biopsied-overnight-cultured embryos and 80% for intact-fresh embryos. Differences between the groups were also apparent in calving rates: 22% for biopsied-cryopreserved control embryos, 31% for biopsied-cryopreserved AC-embryos, 23% for biopsied-overnight-cultured embryos and 63% for intact-fresh embryos. It is concluded that manipulated embryos are associated with lower presumptive pregnancy and calving rates compared with intact-fresh embryos. The highest calving rates for groups of manipulated embryos were achieved in the AC-group. Therefore, addition of AC can be recommended if biopsy is combined with freezing before transfer.

Growth hormone exerts no effect on the timing of the first zygotic cleavage in cattle.

Most of the protocols used for oocyte and embryo quality assessments are invasive and thus reduce embryo viability. Special interest has recently been placed on a search for non-invasive markers related to embryo quality. The characteristics of a non-invasive marker are met by the timing of the first zygotic cleavage (FZC). Therefore, the aim of the present study was to investigate whether growth hormone added to IVM medium influences the timing of the FZC in cattle and also the quality of resulting blastocysts. The novelty of this manuscript concerns two findings: 1) no effect of GH supplementation to IVM medium on the timing of the first zygotic cleavage in cattle, and 2) differences in the relative transcript abundance in bovine day 7.5 blastocysts derived from early and late cleaved zygotes. Cumulus-oocyte complexes aspirated from slaughterhouse ovaries were matured in TCM199 medium supplemented with growth hormone (GH+ 100 ng/ml, GH- control), inseminated, and cultured in sequential media for 7.5 d. Embryo selection was done at 30 hpi (early cleavers EC) and 48 hpi (non-early cleavers NEC). The blastocyst quality was assessed by the total and ICM:TE cell counts, dead cell index, and relative transcript abundance (RA) of five genes affecting embryo development (p66(shc), bax, bcl-2, survivin, Hsp 70.1). The results of this study showed that although GH added to the IVM medium significantly improved the quality of blastocysts on day 7.5 pi, it had no effect on the timing of the first zygotic cleavage expressed by the rate of EC zygotes. The quality of the four categories of blastocysts investigated in this study can be ranked as follows: GH+ EC, followed by GH+ NEC, and further by the GH- EC and GH- NEC embryos. It has to be mentioned, however that the quality of blastocysts derived from the NEC zygotes was significantly improved by the GH supplementation. This is particularly relevant, since those blastocysts were very few and usually characterized by an impaired morphology (e.g., presence of fragmented blastomeres, smaller size).

Impact of in vitro fertilization of bovine oocytes with sex-sorted frozen-thawed spermatozoa on developmental kinetics, quality and sex ratio of developing embryos.

We studied whether bovine embryos developing after in vitro fertilization (IVF) with sex-sorted spermatozoa differed in developmental kinetics, quality and sex ratio from embryos produced with unsorted spermatozoa. Abattoir-derived oocytes were fertilized with X-sorted, Y-sorted or unsorted spermatozoa from a single bull. To evaluate economical use of the sex-sorted spermatozoa, washed spermatozoa from a single straw (2 million spermatozoa) were used to fertilize each batch of collected oocytes without any further isolation steps. Concentration of the unsorted spermatozoa was adjusted accordingly. Fertilizations were assessed by staining sperm asters at 10 hpi and pronuclei at 20 hpi. Embryo development and morphological quality were monitored on days 2, 7, 8 and 9 of the development (IVF = day 0). All embryos were sexed using PCR. Following fertilization, penetration and subsequent cleavage rates were compromised in the X-sorted group compared with the Y-sorted and unsorted groups (penetration: 58.0% vs. 89.8% and 90.0%, cleavage: 65.3% vs. 81.5% and 75.0%). The use of the sex-sorted spermatozoa did not, however, reduce the proportion of transferable embryos (sex-sorted 29.6% vs. unsorted 27.7%) or their quality (quality 1: sex-sorted 36.0% vs. unsorted 19.9%). The Y-sorted spermatozoa produced more transferable embryos of better quality than the X-sorted spermatozoa (days 7-8: 31.9% vs. 26.4%, quality 1: 38.9% vs. 30.6%). On average, out of 10 transferable embryos, nine were of the predicted sex in the X- and Y-sorted spermatozoa groups. These results indicate that low numbers of X- and Y-sorted spermatozoa can be used successfully for female and male embryo production in vitro.

Effects of serum-free in vitro maturation of bovine oocytes on subsequent embryo development and cell allocation in two developmental stages of day 7 blastocysts.

Maturation of oocytes and the subsequent outcome of the in vitro production (IVP) are affected by the composition of in vitro maturation (IVM) medium. To determine the use of serum interfering with effects of single molecules, we aimed at developing simplified IVM medium. The experimental IVM media were: (1) M199-medium supplemented with hormones and serum (control), (2) as 1 but serum was substituted with fatty acid-free serum albumin (FAFBSA) and (3) M199-medium without hormonal and serum supplementation (M199). The quality of embryos was assessed on day 7 by morphology and cryotolerance, as well as by Terminal deoxynucleotidyl Transferase Biotin-dUTP Nick End Labeling (TUNEL) and differential staining. Results showed that the nuclear maturation was suppressed in M199 group alone. Embryo cleavage and development rates, and the proportion of quality 1 blastocysts were lower in the FAFBSA and M199 groups compared to the control. Differences in the cell allocation of fresh embryos were observed at the blastocyst stage, but not at the expanded blastocyst stage. The control group blastocysts had larger number of cells allocated to the inner cell mass (ICM), and the FAFBSA group blastocysts larger apoptotic cell proportion compared to the blastocysts derived from other groups. After cryopreservation, the reduction of ICM proportion and increase of apoptotic cell proportion of embryos were equal between the experimental groups. In conclusion, exclusion of serum from the IVM media reduces embryo development and may cause perturbations in blastocyst development. Differences in the cell allocation of blastocysts between IVM media may appear only when the developmental stages are taken into account.

Copy number variation of testis-specific protein, Y-encoded (TSPY) in 14 different breeds of cattle (Bos taurus).

Multi-copied gene families are prevalent in mammalian genomes, especially within the Y chromosome. Testis specific protein Y-encoded (TSPY) is present in variable copy number in many mammalian species. Previous studies have estimated that TSPY ranges from 50-200 copies in cattle. To examine TSPY localization on the Y chromosome we employed fluorescence in situ hybridization (FISH) and fiber-FISH. The results show a strong signal on the short arm of the Y chromosome (Yp). To investigate TSPY copy number we used relative real-time polymerase chain reaction (PCR) to analyze the DNA of 14 different cattle breeds. Variation both within and between breeds was observed. All breeds show significant variation in TSPY copy number among individual members. Brown Swiss (161 copies, CI = 133-195) had higher average levels of TSPY and Western Fjord Cattle (63 copies, CI = 45-86) had lower levels than some breeds. Overall, however, most breeds had a similar average TSPY copy number. The pooled average was 94 copies (CI = 88-100). The significance of the TSPY array remains uncertain, but as the function of TSPY is unraveled the purpose of the array may become clearer.

Embryo production from superovulated Holstein-Friesian dairy heifers and cows after insemination with frozen-thawed sex-sorted X spermatozoa or unsorted semen.

The aim of this study was to evaluate embryo production in superovulated Holstein-Friesian dairy heifers and cows inseminated with either X-sorted spermatozoa (2 million/dose) or unsorted semen (15 million/dose). Experiment 1 at the research farm involved eight heifers, six cows and semen of one Holstein bull. All transferable embryos were diagnosed for sex. Experiment 2 included embryo collections on commercial dairy farms: X-sorted spermatozoa from three Holstein bulls were used for 59 collections on 28 farms and unsorted semen from 32 Holstein bulls were used for 179 collections on 79 farms. Superovulations were induced by eight declining doses of FSH (total of 12 ml for heifers and 19 ml for cows) starting on days 8-12 of the estrus cycle. Inseminations began 12h after the onset of estrus and were performed two to four times at 9-15 h intervals. Low-dose X-sorted inseminates were deposited into uterine horns and unsorted semen was placed into the uterine body. In Experiment 1, on average 70.3 and 75.0% of embryos recovered from heifers, and 48.4 and 100% of embryos recovered from cows were of transferable quality in X-sorted and unsorted groups, respectively. The proportion of transferable female embryos produced approximately doubled when insemination was with X-sorted spermatozoa compared to insemination with unsorted semen (heifers 96.4% versus 41.1%; cows 81.1% versus 39.8%). In Experiment 2, estimated 53.9 and 65.5% of embryos recovered from heifers, and 21.1 and 64.5% of embryos recovered from cows were of transferable quality in X-sorted and unsorted groups, respectively. Proportions of unfertilized oocytes were 21.1 and 10.6% for heifers and 56.0 and 14.4% for cows in X-sorted and unsorted groups, respectively. Consequently, cows inseminated with X-sorted spermatozoa produced significantly smaller proportions of transferable embryos (p<0.005) and significantly larger proportions of unfertilized oocytes (p<0.001) than those inseminated with unsorted semen. Proportions of quality 1 or degenerated embryos were similar for the two treatments in both heifers and cows. Within treatments, bulls did not significantly affect the proportions of transferable, unfertilized or degenerated oocytes/embryos. It was concluded that using low-dose X-sorted spermatozoa rather than normal-dose unsorted semen for the insemination of superovulated embryo donors can improve the proportion of transferable female embryos produced but this potential may not be achieved in commercial practice, particularly in cows, because of reduced fertilization rates when using low doses of X-sorted spermatozoa.

Gene expression and protein distribution of leptin and its receptor in bovine oocytes and preattachment embryos produced in vitro.

Many investigations point out the important role of leptin during the preimplantation development. Transcripts for the leptin gene (LEP) and its receptor (LEPR) have been identified in several tissues related to reproduction (e.g. ovaries, testis and oviduct) in both human and mouse. This work shows for the first time the expression and distribution patterns of LEP and LEPR in bovine oocytes and in vitro-produced embryos. Gene expression was analysed by reverse transcription PCR and real-time PCR, and the proteins were localised by immunostaining. This study included immature and mature oocytes, zygotes, two-, four-, eight- to 16-cell embryos, morulae and blastocysts and the LEP transcript was identified throughout all stages of bovine preimplantation development. However, mRNA for the LEPR gene was detected at all stages, excluding four-cell embryos. Expression of both LEP and LEPR genes was reduced at the eight- to 16-cell stage. This in addition to the absence of LEPR mRNA in four-blastomere embryos may suggest that maternally derived transcripts degenerate towards the eight- to 16-cell stage coinciding with embryonic genome activation at eight- to 16-cell stage and subsequent appearance of embryonic mRNA. Immunofluorescent staining demonstrated that LEP and LEPR proteins form a spherical rim beneath the oolemma. After maturation, however, the proteins became evenly distributed within the cytoplasm. In two- to eight-cell embryos, fluorescence was observed in the apical surface of the blastomeres, and from 10- to 16-cell stage in the apical region of outer blastomeres. This pattern persisted to the blastocyst stage, leading to LEP and LEPR distribution within trophoblast cells, but not in the inner cell mass. These results support previous findings on polar distribution of proteins within mammalian oocytes and embryos, as well as suggests leptin's potential role during early mammalian development and implantation.

Maturation medium supplements affect transcript level of apoptosis and cell survival related genes in bovine blastocysts produced in vitro.

The aim of the present study was to investigate whether protein or macromolecule supplements to in vitro maturation media affect transcript abundance of seven genes (Bax, Bcl2, Hsp70, IGF1, IGF1R, IGF2, and IGF2R) in oocytes and blastocysts. Cumulus-oocyte complexes aspirated from slaughterhouse ovaries were matured in TCM199 medium supplemented either with 10% FBS, 6% fatty acid free BSA (fafBSA) or 4% PVP40, then inseminated and cultured in vitro for 9 days. Transcript abundance analysis was carried out on immature and in vitro matured oocytes, as well as on blastocysts. Total RNA was isolated from pools of oocytes and embryos, reverse transcribed into cDNA and subjected to transcript analysis by real-time PCR. No transcript of IGF1 gene was detected either in oocytes or in blastocysts. Maturation conditions significantly affected transcript levels of investigated loci in blastocysts but not in matured oocytes, with one exception. Only relative abundance (RA) of IGF2 gene was higher in oocytes matured with fafBSA. Moreover, oocyte maturation with fafBSA elevated transcript abundance of IGF1R, IGF2, and IGF2R genes in resulting blastocysts, whereas Hsp70 transcription was stimulated by FBS supplementation. Thus, under described conditions, fafBSA may be the optimal supplement to IVM medium due to higher transcript level of growth factor coding genes accompanied by a lower transcript level of Hsp70.

Supplements to in vitro maturation media affect the production of bovine blastocysts and their apoptotic index but not the proportions of matured and apoptotic oocytes.

The objective of this study was to compare the effect of different supplements to the basic IVM medium (TCM199) on the efficiency of cattle oocyte maturation and blastocyst production, and the incidence of apoptosis in both oocytes and blastocysts. Two protein supplements (FBS and fafBSA) and a macromolecule (PVP40) were compared in a 3 treatmentsx9 replicates design. Cumulus-oocyte complexes (COCs) aspirated from slaughterhouse ovaries were matured for 24h in TCM199 medium supplemented with 10% FBS, 6% fafBSA or 4% PVP40 (50-70 COCs in each treatment/replicate), then inseminated and cultured in vitro for 8 days. Immature and mature oocytes as well as Day 8 blastocysts were subjected to TUNEL analysis. Cleavage rate was monitored on Day 2 post-insemination (pi), whereas blastocyst yield on Day 8 pi. The composition of maturation media did not affect zygotic cleavage rate on Day 2 (on average 71.0%), however the blastocyst rate on Day 8 pi was significantly lower (P<0.001) for embryos derived from oocytes matured with PVP40 (16.0%) than for those matured with FBS (22.4%) or fafBSA (22.1%). The rate of TUNEL positive oocytes differed significantly between immature (1.4%) and mature (11.2%) oocytes (P<0.01). Supplements to maturation medium were not related to the incidence of apoptosis in mature oocytes (11.2%) and the rate of oocytes at the second metaphase stage (71.5%). Cumulus cell expansion was reduced by maturation in medium supplemented with PVP40. This macromolecule was also correlated with higher apoptotic index in blastocysts (5.8%) when compared to FBS (3.2%) and fafBSA (3.1%; P<0.001). In conclusion, lower blastocyst rate and elevated apoptotic index in embryos derived from oocytes matured with PVP40 may suggest that synthetic macromolecule provides less balanced environment for oocyte maturation and therefore should be treated with caution.

Respiration rates of individual bovine in vitro-produced embryos measured with a novel, non-invasive and highly sensitive microsensor system.

Oxygen consumption is a useful parameter for evaluating embryo quality, since it provides a valuable indication of overall metabolic activity. Over the years, several approaches have been used to measure the respiration rates of individual embryos, but a convincing method has not yet been reported. In this study, we introduce and have validated a novel high resolution microsensor technology to determine the respiration rates of individual embryos at different developmental stages. We have employed this technology to investigate the correlation between respiration rate and embryo morphology, diameter and sex. Following morphological evaluation, individual respiration rates of day 3 (n = 18) and day 7 (n = 60) bovine in vitro-produced embryos were determined. Of the measured embryos, 64 were lysed for sex diagnosis by PCR. Average respiration rates of day 7 embryos (1.30 +/- 0.064 nl/h) were 3.4-fold higher than day 3 embryos (0.38 +/- 0.011 nl/h). On day 7, the average respiration rate of quality 1 blastocysts was significantly higher than the respiration rates of the lower qualities. For both day 3 and day 7 embryos, respiration rates were directly influenced by embryo diameter but did not differ between sexes. These results have demonstrated that the novel microsensor technology can be used to accurately and rapidly (8 min) measure the respiration rates of individual embryos at different developmental stages. Respiration rates were only in partial agreement with embryo morphology, suggesting a slight discrepancy between these two methods in assessing embryo quality. It is likely that a combined assessment of embryo respiration and morphology would improve embryo classification and subsequent selection.

Sex-chromosome linked gene expression in in-vitro produced bovine embryos.

The expression of XIST, G6PD, HPRT, ZFX and ZFY were investigated in in-vitro produced bovine embryos. Transcripts of these genes were assayed by RT-PCR in pools of pre-compaction stage embryos and sexed pools of morulae and blastocysts. The expression of XIST, G6PD, HPRT and ZFX in female and male morulae and blastocysts were compared using a semi-quantitative RT-PCR. G6PD, HPRT and ZFX transcripts were noted in all pre-compaction stage embryos and in female and male blastocysts. ZFY transcripts were detected in unsexed pools of 8-16-cell stage embryos and in male blastocysts. XIST transcripts were detected in unsexed pools at the 8-16 cell stage, in male and female morulae, and in female blastocysts. The level of XIST RNA was significantly higher in female morulae than in males. Levels of G6PD and HPRT RNA were also higher in female morulae and blastocysts than in males, but only G6PD levels were significantly different between the sexes. The expression of ZFX was also significantly higher in female than in male blastocysts. These results show sexually dimorphic expression of sex chromosome linked genes prior to the blastocyst stage in in-vitro produced bovine embryos.

Sex determination of bovine embryo blastomeres by fluorogenic probes.

One of the major challenges of using genetic information in marker assisted selection (MAS) is the detection of multiple marker loci from a small biopsy sample of a preimplantation stage embryo. The objective of this study was to develop a fast, nested, multiplex preamplification, polymerase chain reaction (PCR) method for the determination of sex in bovine embryo blastomeres. For this aim, ZFX/ZFY sequences were preamplified simultaneously with other genomic regions. The preamplification product was used as a template in an allelic discrimination assay, with nested primers and sex specific fluorogenic probes for ZFX and ZFY. Fluorogenic probes were used to eliminate the need for time consuming electrophoresis. Compared to sexing with Bovy/kappa-casein co-amplification method and other replicates from the same embryo, the accuracy of sexing with the use of fluorogenic probes after preamplification was 99% (112/113 blastomeres). The amplification efficiency was 96% (113/117 blastomeres).

Sex-linked genes are not silenced in fetal bovine testes expressing X-inactive specific transcript (XIST).

X-inactive specific transcript (XIST), which is thought to be the central factor for the X-inactivation process in female mammals, is known to be expressed in males during spermatogenesis. Our studies have shown that XIST is not only expressed in adult bovine testis but is also expressed in fetal, newborn, and prepubertal testes long before spermatogenesis is established. To determine whether the XIST expressed in fetal testes is involved in silencing the genes on the X chromosome, we investigated the status of X-linked genes, including glucose-6-phosphate-dehydrogenase (G6PD), hypoxanthine phosphoribosyl transferase (HPRT), and X-linked zinc finger protein gene (ZFX), in fetal bovine gonads at the developmental stage, when meiosis is initiated in fetal ovaries in this species. Reverse transcription and a semiquantitative polymerase chain reaction based on the optical density of each gene-specific band relative to that of the co-amplified Quantum RNA 18S Internal Standard (Ambion, Austin, TX) showed that the XIST gene was expressed in the testes of approximately 90-day-old fetuses and was silent in all their nongonadal organs tested, although at a significantly lower level than that in fetal organs of female fetuses. Our observation that the expression of X-linked genes in the fetal testis was comparable to that in male nongonadal organs, in which X inactivation does not occur, indicates that the low level of XIST, or XIST-like RNA, expressed in the fetal bovine testis is not involved in silencing X-linked genes.

Developmental kinetics of in vitro produced bovine embryos: the effect of sex, glucose and exposure to time-lapse environment.

In this study, a simple time-lapse video recording system was used to compare developmental kinetics of female and male bovine embryos produced in vitro. Following embryo sex determination, the timing of each cleavage up to the 4-cell stage was compared between the sexes from the videotapes after culture in the presence and absence of glucose. In the second experiment, the consequences of exposure to a time-lapse video recording (TL) environment were studied by culturing embryos further until day 7 in an incubator, followed by collection and sex determination of morulae and blastocysts. In the absence of glucose, female embryos cleaved earlier than male ones. In the presence of glucose, however, male embryos cleaved earlier than female ones. There was no difference in the number of morulae/blastocysts in the absence of glucose, but in the presence of glucose more male than female embryos reached the morula and blastocyst stage. Exposure to the TL environment itself also had a sex-related effect, being more detrimental to male than female embryos. The difference in the number of functional X chromosomes between the sexes during early preimplantation development could explain these findings. In females, an increased capacity for oxygen radical detoxification through the pentose phosphate pathway could result in a reduced cleavage rate. Furthermore, glucose may influence the expression of enzymes located on the X chromosome. According to these results, a simple time-lapse video recording system is suitable for investigating embryo developmental kinetics and perhaps for the selection of embryos with the greatest developmental potential.

Sex related embryo development.

Although sexual dimorphic development in the mammalian embryo prior to differentiation of the gonad has been documented, there are many seemingly conflicting observations and gaps in our understanding of this process. Conditions that influence the process include gamete interaction, that might give one sex and advantage in the fertilization process and in rates of blastomere cleavage that would allow one sex to accumulate cells at a faster rate. In this scenario, males could accumulate more cells within a defined window of development. Another key difference between males and females is the number of copies of genes located on the sex chromosomes. Transcripts from the Y-chromosome are thought to function as transcription factors, which could accelerate development. Conversely, the X-chromosome contains genes that code for rate limiting steps in pathways key to embryo metabolism and stress reduction. It can be envisioned that prior to X-chromosome inactivation in females, elevated levels of transcripts for such genes may enable greater protection from environmental stress and regulate growth. As we gain a better understanding of how males and female develop we will be able to exert greater control over the manipulation of the sex ratio for the offspring of domestic animals.

Successful transfer of biopsied equine embryos.

Embryo biopsy has been used to detect inherited disorders and to improve the phenotype by analyzing of linkages between marker loci and the desired characteristics. Unfortunately, early procedures required the removal of a large portion (one-half) of the embryo for analysis, and the transfer of bisected equine embryos has not been particularly successful. Recent discovery of the polymerase chain reaction (PCR) has made possible the detection of specific DNA sequences from only a few cells. We investigated whether the removal of a small biopsy would allow for successful PCR and normal embryonic development. In the study reported here, 14 microbladebiopsied Day 6 to 7 equine embryos were transferred nonsurgically into recipient mares. The sex of each embryo was determined from the biopsy by means of restriction fragment length polymorphism analysis of the ZFY/ZFX loci after PCR amplification. The embryos were sexed as 8 females and 6 males on the basis of PCR assay results. Two embryos were biopsied using a needle aspiration technique, but no PCR amplification products resulted from these attempts. Eight intact control embryos were transferred to recipient mares using the same method. Pregnancy rates were 3 14 and 6 8 for the microblade biopsy and control groups, respectively. All of the microblade biopsy group pregnancies were females. One was aborted for cytogenetic analysis. Two were born after normal gestation. With improved pregnancy rates, this technique could be used for preimplantation diagnostics of equine embryos. As gene mapping advances and associations between particular DNA sequences and inherited traits become established, a rapid PCR technique could be used to select embryos before transfer.

Sex diagnosis of equine preimplantation embryos using the polymerase chain reaction.

A rapid and reliable method for sex determination of preimplantation-stage equine embryos has not been available. The aim of the present study was to find an enzyme which would distinguish sexes in the horse by finding a polymorphic restriction site between the ZFY and ZFX homologues amplified by the polymerase chain reaction (PCR). Altogether, 38 different restriction enzymes were tested using female and male DNA extracted from blood. The primers used for amplification were selected from conserved sequences between human ZFY and ZFX genes and mouse Zfy-1 and Zfy-2 genes. Nine enzymes cut the PCR product of approximately 450 basepairs, but only Bsm I yielded different banding patterns in female and male DNA. All blood samples were correctly diagnosed. To test the method on embryonic cells, 17 horse demi-embryos were obtained from expanding blastocysts 220 to 950 mum in diameter. Demi-embryos were further cut into 3 to 7 parallel samples which were analyzed individually to test the repeatability of the method. Eight of the original embryos were diagnosed as females and 9 as males. No misidentifications were observed within the embryonic samples, suggesting that this sexing method is highly reliable. This study provides a rapid and accurate method to sex horse embryos.