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One of the most puzzling aspects of monotreme reproductive biology is how they determine sex in the absence of the SRYgene that triggers testis development in most other mammals. Although monotremes share a XX female/XY male sex chromosome system with other mammals, their sex chromosomes show homology to the chicken Z chromosome, including the DMRT1gene, which is a dosage-dependent sex determination gene in birds. In addition, monotremes feature an extraordinary multiple sex chromosome system. However, no sex determination gene has been identified as yet on any of the five X or five Y chromosomes and there is very little knowledge about the conservation and function of other known genes in the monotreme sex determination and differentiation pathway. We have analysed the expression pattern of four evolutionarily conserved genes that are important at different stages of sexual development in therian mammals. DMRT1is a conserved sex-determination gene that is upregulated in the male developing gonad in vertebrates, while DMRT7is a mammal-specific spermatogenesis gene. ATRX, a chromatin remodelling protein, lies on the therian X but there is a testis-expressed Y-copy in marsupials. However, in monotremes, the ATRXorthologue is autosomal. WT1is an evolutionarily conserved gene essential for early gonadal formation in both sexes and later in testis development. We show that these four genes in the adult platypus have the same expression pattern as in other mammals, suggesting that they have a conserved role in sexual development independent of genomic location. [ABSTRACT FROM AUTHOR]

The article presents abstracts on bovine and equine sexing topics which include producing female bovine embryos in vitro using X-chromosome-selected sexed semen, a procedure for genomic evaluation of bovine embryos to determine gender and hereditary defects within 24 hours after collection, and sex determination of equine embryos by polymerase chain reaction using blastocoele fluid.

The article presents abstracts on sexing which include a method of processing sex-sorted bovine sperm that improve conception rates and a method of processing sex sorted bovine sperm that improves post-thaw sperm quality and in vitro fertility.

Embryo-splitting technology provides an effective procedure for increasing the number of transferable embryos per donor, producing genetically identical offspring and facilitating embryo sexing. The ability to identify the sex of embryos before transfer will offer a reliable, economical and practical procedure for buffalo breeding. In this study, we have assessed the feasibility of production of offspring with controlled sex in buffalo by first comparing the effect of blastocyst quality on the viability of demi-embryos and then identifying the sex of a demi-embryo by multiplex-nested PCR before transfer into the recipient. In vitro-matured buffalo oocytes were fertilized by IVF and cultured to the blastocyst stage for 6 to 7 days as described by Lu et al. (2007 Anim. Reprod. Sci. 100, 192-196). These blastocysts were classified in terms of their developmental pattern and morphology on a scale of 1 to 3 grades as described by McEvoy et al. (1990 Theriogenology 33, 1245-1253). Blastocysts were split into 2 equal parts by a micromanipulation system. Viability of the resulting demi-embryos was confirmed by formation of a blastocoel cavity and definite inner cell mass after culture for 24 h. One of the zone-free demi-embryos derived from a grade-1 blastocyst was cultured in TCM 199 supplemented with 10% fetal bovine serum for another 2 h, then was transplanted to a spontaneous oestrous recipient. The other demi-embryo was used for sexing by multiplex-nested PCR (Fu et al. 2007 Theriogenology 68, 1211-1218). The results showed that grade-1 blastocysts yielded more viable demi-embryos than grade-2 and grade-3 blastocysts [P < 0.01; 73/92 (79.67%) vs 32/76 (47.05%) vs 26/94 (26.53%), respectively]. Transplantation of the presumed-Y demi-embryo derived from grade-1 blastocyst into a recipient resulted in the birth of a male buffalo calf. To the best of our knowledge, this is the first buffalo calf produced following embryo splitting and PCR sexing of the embryo at the blastocyst stage. Successful birth of the desired-sex offspring in the present study indicates the feasibility of using embryo splitting in combination with multiplex-nested PCR sexing to produce offspring of controlled sex in swamp buffalo. However, the quality of embryos before splitting was an important factor governing the in vitro development of viable demi-embryos. This study was supported by the Guangxi Science and Technology R&D Program (0626001-3-1, 0815008-2-4). [ABSTRACT FROM AUTHOR]

Cell sorting is an important part of the sperm sexing process. The objective of this study was to compare the efficiency of in vitroembryo production using sexed frozen–thawed bull sperm sorted with 2 types of cell sorter. Ejaculates from 2 Bos taurus(Holstein, 5 years old) bulls underwent conventional processing (control) or sorting for X chromosome bearing sperm using MoFlo®SX (SX, Dako, Fort Collins, CO, USA) or MoFlo®XDP-SX (XDP, Beckman Coulter, Fullerton, CA, USA) following XY™ sperm-sorting protocols. Processed sperm samples were cryopreserved in 0.5-mL plastic straws. Cumulus–oocyte complexes obtained from abattoir-derived ovaries were matured for 20h in HEPES–TCM-199 (Lu and Seidel 2004 Theriogenology 62, 819–830) and randomly assigned to each of 3 sperm treatment groups. Thawed sperm were centrifuged for 20min at 448×gthrough an ISolate®(Irvine Scientific, Santa Ana, CA, USA) gradient (45:90%). Sperm pellets were washed in IVF100 (Hoshi 2003 Theriogenology 59, 675–685) by centrifugation for 5min at 252×g. Oocytes were co-incubated with washed sperm (5 to 10×106sperm mL-1) in IVF100 (Hoshi 2003 Theriogenology 59, 675–685) for 8h at 38.5°C in 5% CO2and 95% air (Day 0). Presumptive zygotes were cultured for 90h in CDM-1 (Lu and Seidel 2004 Theriogenology 62, 819–830) and then washed and cultured in IVD101 (Hoshi 2003 Theriogenology 59, 675–685) at 38.5°C in 5% CO2, 5% O2, and 90% N2. Cleavage rates on Day 2 and blastocyst rates on Day 7 to 9 were recorded after insemination. Two-way ANOVA was used for data analysis, followed by Fisher’s PLSD test. Experiments were replicated 4 times for bull A (total of 1350 oocytes used) and 5 times for bull B (total of 1529 oocytes used). The data are summarised in Table 1. No interaction was observed between the treatments and bulls. Cleavage rates were not significantly different in the 3 treatment groups. However, blastocyst rates were significantly lower in both SX (P0.8). Bull B showed significantly poorer results than bull A regarding both cleavage (P<0.003) and blastocyst (P<0.02) rates. MoFlo®SX (analogue processing) has been used for a decade, and XDP (digital processing) is the replacement model with its accelerated sorting speed. The current results indicated that the in vitroembryo production efficiency did not differ between sperm sorted with either SX or XDP. We suggest that sperm can be sorted using XDP without compromising sperm health. Table 1.Cleavage and blastocyst rates after IVF with 2 Holstein bulls for three sperm treatments [ABSTRACT FROM AUTHOR]

The use of sexed semen in dairy cattle almost eliminates the birth of unwanted male calves and can lead to increased milk production by means of genetic selection exerted on the females. However, AI with sexed semen still presents several obstacles to overcome. Holstein-Zebu cows are responsible for 80% of the milk produced in Brazil. These crossbred animals are used extensively throughout the country because their physiologic and morphologic characteristics are compatible with milk production in the tropics which results in an economic performance advantage. The objective of this study is to evaluate the conception rates with sexed semen, comparing it to the use of non-sorted semen in crossbred dairy cows. In addition, two insemination times had been studied to determine the best timing for AI with the sexed semen. Both studies were carried out in the Experimental Station Seropedica of Pesagro-RJ, located in the state of Rio de Janeiro, Brazil. The experiment was done in 2008/2009 (January to April). In the first Trial 56 Holstein-Zebu females, composed of 1/2 to 7/8 Holstein genetics, were inseminated 12 h after estrus detection with non-sorted semen (control group n= 29, 14 heifers and 15 cows) or with sexed semen (experimental group n= 27, 13 heifers and 14 cows). The semen utilized was from the same bull and thawing was performed intravaginally. In the second trial, 55 females were inseminated only with sexed semen from the same bull, with thawing in water at 35°C, at two different times: 12 h after estrus observation (control group n= 27, 12 heifers, 4 primiparous, and 11 multiparous) and 15 h after estrus observation (experimental group n= 28, 12 heifers, 5 primiparous, and 11 multiparous). The pregnancy diagnoses were done by rectal palpation and transrectal CAT scan at Day 45. Statistical analyses had been done using Chi-square. The conception rate in the first trial was of 17.2% (5/29) for sexed semen and 53.8% (14/26) for non-sorted semen (P 0.05). It is possible that one of the causes of fertility reduction with sexed semen is that sexed-sperm remain viable for a shorter time after the insemination. Inseminations with sexed semen done 15 h after estrus detection, which can be more synchronized with the time of ovulation, and when accompanied by closely controlling the thawing process, show results similar to those obtained with conventional semen. [ABSTRACT FROM AUTHOR]

Sex-dependent differences in mammalian embryo phenotype are apparent at the preimplantation stage of development, before the appearance of sex-specific cells. The ratio of male:female embryos may be modified by environmental factors such as maternal diet in vivoand the composition of embryo culture media in vitro.We have used amino acid profiling (AAP), a defined, non-invasive metabolic marker of developmental potential to compare the effect of sex on the metabolism of bovine preimplantation blastocysts and expanded blastocysts conceived in vivo(n= 35) or produced in vitro(n= 172). Blastocysts were incubated individually for 24 h in synthetic oviduct fluid medium plus a close-to-physiological mixture of amino acids. The depletion or appearance of 18 amino acids was measured using high-performance liquid chromatography. Blastocysts were then sexed by PCR and the outcome related to AAP. Amino acid depletion by in vitro-produced blastocysts was higher than in embryos conceived in vivo(P= 0.02). Net appearance of amino acids was higher in the medium from early blastocysts produced in vitro(P= 0.018) although this rise was lost at the expanded stage. There were marked differences in the amino acid profiles of male and female embryos produced in vitro: female embryos exhibited significantly increased depletion of arginine, glutamate, and methionine and appearance of glycine, while male embryos displayed increased depletion of phenylalanine, tyrosine, and valine. Overall, in vitro-produced blastocysts exhibited gender-specific differences in metabolic profiles of 7 out of 18 amino acids; in vivo-produced blastocysts exhibited differences in 2 out of 18 amino acids. These differences had disappeared by the expanded blastocyst stage. Our experiments reveal striking differences in the metabolism of preimplantation embryos conceived in vivoand in vitro, some of which, particularly in the case of the in vitro-produced embryos, are dependent on embryo sex. Moreover, in vivo-derived embryos tend to have a reduced metabolism consistent with the Quiet Embryo Hypothesis, which proposes that higher quality embryos have less molecular and cellular damage than those of a lower quality and thus have a reduced need to take up nutrients for repair processes. Supported by a Wellcome-VIP/University of York Fellowship to RGS. [ABSTRACT FROM AUTHOR]

The aim of this study was to test a FISH approach using ovine-painted probes specific for the chromosomes X and Y, on goat interphase and metaphase nuclei of blastocyst cells. Oocytes of prepubertal goats were recovered at a slaughterhouse and selected by morphological criteria. Oocytes were matured in TCM-199 supplemented with hormones, serum and cysteine at 38.5°C for 27 h. IVM-oocytes were fertilized in vitroand the presumptive zygotes were cultured for 10 days in SOF with 10% FCS at 38.5°C, 5% CO2and 5% O2. The blastocyst nuclei were spread using a modified Tarkowski method (1966). Briefly, individual embryos were immersed into hypotonic solution for 5 min, followed by fixative solution of methanol/acetic acid (Carnoy''s solution) until the embryos acquired a transparent appearance. Next, the embryos were transferred to a Superfrost plus Slide (Menzel Gläser, Braunschweig, Germany) in a small droplet mixture of distilled water and Carnoy The zona pellucida and the blastomere cytoplasm dissolved gradually and Carnoy solution was added dropwise to the slide before the nuclei dried out. The morphology and total number of nuclei in each embryo were analyzed under phase contrast microscope and stored at -18°C. Embryos with appropriate fixation were then subjected to hybridization with ovine-painted probes specific to chromosomes X (Fluorocrom green-FITC) and Y (Fluorocrom orange-TAMRA) (Chrombios-Molecular Cytogenetics GmbH, Raubling, Germany) according to the manufacturers protocol and adjusted for the caprine species. Briefly, slides were then incubated at 60°C for 1 h. The chromosomal DNA was then denatured by immersing slides in 70% formamide/30% 2 × SSC at 70°C for 1.5 min, and immediately dehydrated in an ethanol series (70%, 90%, and 100%), 4 min duration per solution and air dried. In parallel, X- and Y-probes were added to the hybridization solution (50% deionized formamide, 10% dextran sulfate, 2 × SSC) and denatured at 75°C for 10 min. Aliquots (0.5 to 1.5 μL) of this solution were placed on each slide and sealed with a coverslip and glue prior to incubation at 37°C (Hybrite; Vysis Inc, Dowers Grove, IL, USA). After 22 to 24 h the coverslip was removed and the slides were washed three times. The first and third washes were performed with 2 × SSC at room temperature while the second wash was in a 0.4 × SSC/0.1% Tween at 73°C for 3 min. Nuclear DNA was counterstained with diamino-phenyl-indole solution (DAPI) and examined with a fluorescence microscope (Olympus BX61, Olympus America Inc., Melville, NY, USA) equipped with appropriate filters. From a total of 69 blastocysts, 11 355 blastomeres were analyzed and 7,825 were correctly hybridized (68.9%). The results of the embryo sexing were: 24 embryos XX, 11 XY, 22 polyploid embryos (of which 13 presented more than 80% of cells XX), 3 haploid embryos (X0), 2 tetraploid embryos, and 5 no result. In summary, goat blastocysts were successfully sexed using FISH with painted ovine X- and Y-specific probes. Grant sponsor Spanish Ministery of Science and Innovation.Code: AGL2007-60227-CO2-01. [ABSTRACT FROM AUTHOR]

The objective of this study was to optimize bovine sperm storage for up to 20 h between semen collection and sex sorting followedby cryopreservation. Two successive ejaculates were obtained from mature dairy bulls (Holstein, n= 5; Jersey, n= 3) via artificial vagina. Treatments were then applied to the neat semen to which antibiotics were added as recommended by Certified Semen Services (Columbia, MO). Nothing further was added to the control samples until staining with Hoechst 33342 for sorting. For Treatment 1, semen was diluted 9:1 with a MOPS solution resulting in 24 mMMOPS and similarly, Treatment 2 resulted in 24 mMMOPS +2% egg yolk. A subsample of each treatment and control was sorted by flow cytometry shortly after collection, and sperm then were frozen following standard processing procedures. The other subsample was stored at 15-18°C and sorted 20 h after collection followed by cryopreservation. pH measurements were made before staining samples for sorting. Samples were evaluated post-thaw for subjective progressive and total sperm motility, by computer-assisted sperm analysis (CASA, Berkeley, CA, USA), and by flow cytometry for sperm viability using propidium iodide and SYBR-14. Treatment 1 performed better than the control (Table 1), while results for Treatment 2 were similar to the control. Second ejaculates were superior to first ejaculates. pH measurements showed that addition of MOPS kept the pH about 0.2 units higher than the control, but pH declined similarly over time in all groups. While responses for the 20 h sort were numerically lower than the 0 h sort (P> 0.1), the majority of responses were acceptable for most, but not all bulls. In conclusion, storing sperm in 24 mMMOPS was beneficial. Surprisingly, 2% egg yolk negated the beneficial effect of MOPS, possibly due to increasing osmolarity by ∼15mOsM/kg due to pH adjustment. Addition of MOPS provided better results than the control for both the 0 h and 20 h sorts. Table 1.Main effect means of semen characteristics [ABSTRACT FROM AUTHOR]

Within an in vitroproduction system, bulls differ with respect to their semen potential in generating embryos when the variables of maternal effects are minimized (Marquant-le-Guienne and Humblot 1992 Ann. Zootech. 41, 361-370). We have tested the hypothesis that even with this variation among bulls, there is also an intra-bull variation among frozen sexed semen batches (Sexing Technologies, Navasota, TX, USA; personal communication) when used with IVF. In an embryo commercial production system, 5058 viable oocytes obtained by ovum pick-up with ultrasound from 193 Nelore cows (Bos indicus) over a 12-month period were matured in vitrofor 24 h in TCM-199 (Gibco, Life Technologies, Carlsbad, CA, USA) containing 0.5 μg mL-1FSH (Bioniche Animal Health, Belleville, Ontario, Canada), 50 μg mL-1LH (Intervet, Boxmeer, the Netherlands) and 10% fetal bovine serum (Gibco). Mature oocytes were inseminated in vitrofor 18 h in IVF-Talp medium (BSA-FAF 6 mg mL-1; 10 ng of heparin, Sigma, France), using 3 different batches (I, II, III) of frozen-thawed sexed semen from 4 bulls (A, B, C, D), separated with a Percoll gradient (45:90; Sigma, St-Quentin Fallavier, France). Putative zygotes surrounded in cumulus cells were transferred in CR2aa medium droplets (Rosenkrans and First 1994 J. Anim. Sci. 72, 434-437) with 3 mg mL-1BSA-FV (Sigma) for 163 h in a humidified incubator at 39°C, with an atmosphere of 5% CO2in air. Total number of oocytes, total number of blastocysts, and embryonic developmental rates for each bull and respective batch are reported in Table 1. The chi-square test was measured with a significance level of P< 0.05 and showed that there is difference between the batches used with respect to developmental rates of blastocysts. Therefore, there is intra-bull variation in the ability to produce in vitroembryos according to the batch of frozen sexed semen. Table 1.Viable oocytes (VO), total blastocysts (TB), and embryo development rate (%E) by bull and batch used in IVF [ABSTRACT FROM AUTHOR]

An abstract of the study "Bovine Embryo Sexing in Field Conditions: Efficacy of the Polymerase Chain Reaction Method and Pregnancy Rates in Dairy Herds Located in the South and Southeast Regions of Brazil," by R. H. Alvares and colleagues is presented.

An abstract of the study "Use of Doppler Ultrasound for Equine Fetal Sex Determination," by H. L Resende and colleagues is presented.

Embryo transfer (ET) in cattle speeds up genetic gains, but its use is limited by cost-benefit analysis. In addition other techniques can be developed, such as sexing and bipartition of embryos. An alternative to improve the economic viability of ET in cattle would be to bipartition embryos in order to increase the yield of the technique. The aim of this study was to investigate the operational viability of embryo bipartition technique in an ET program in cattle and to study aspects related to the viability and development of bovine hemi-embryos (HE) compared with intact ones. The embryos were collected by nonsurgical technique 7 days after the onset of oestrus. Viable structures (130) from 49 embryo collections from 25 Aberdeen and Simmental donor cows and heifers were used. Only embryos in compact morula, early blastocyst and blastocyst stage, with a morphological range from excellent to good grade (IETS Grade 1), were split. The embryos, split (78) or not (52), were transferred into the uterine horn ipsilateral to the corpus luteum of the recipients 7 days after oestrus. The treatment groups evaluated were T1 (intact embryo, n = 52), T2 (1 HE, n = 27) and T3 (2 HE, n = 51). Crossbred heifers were used as recipients and pregnancy diagnosis was done at 60 to 80 days of gestation. The embryos of T1, T2 and T3 were classified morphologically as excellent or good and by developmental stage as morula, early blastocyst, or blastocyst, distributed as follows: T1: 30, 22 and 16, 17 and 19; T2: 15, 12 and 7, 9 and 11; and T3: 29, 22 and 15, 15 and 21, respectively. The birth rate per original embryos was greater for T2 than for T1 and T3 (Table 1). The pregnancy rates for excellent and good embryos and morulae, early blastocysts and blastocysts were not different (P > 0.05). The T1, T2 and T3 twin births were 0, 1 and 5, respectively. It is concluded that embryo bipartition technique applied in a commercial ET program is a viable operational technique. Table 1. Pregnancy at 60 to 80 days and birth rate per original embryos used Supported by CAPES, CNPq, FAPEMIG and BIOTRAN. [ABSTRACT FROM AUTHOR]

Flow cytometric seperation of X- and Y-chromosme-bearing sperm is the only reliable technique that has been used succesfully for commercialization of sexed semen in the dairy industry. However, sperm sexing results in lower fertility compared to conventional semen. Due to logistics like the distance between bull station and sorting laboratory, the number of flow cytometers, and the time needed for the sorting process itself, the holding time between ejaculation and freezing of sex-sorted semen is variable. This holding time could influence the fertility of sex-sorted semen. The aim of this study was to evaluate the effect of different holding times before sexing on the non-return rates on Day 56 post insemination (NRR56). Following collection, raw ejaculates were transported at room temperature (19°C) from two semen collection centers to the central sorting laboratory. Within 2-4 h after collection, volume, sperm concentration and quality of the ejaculates were determined. When accepted, aliquots of 1 mL of semen were stained every 45-60 min for 2 h. Subsequently, sexing was performed according to procedures described previously, using MoFlo SX™ sperm sorters (Garner DL and Seidel GE 2008 Theriogenology 69 : 886-895). Each aliquot of 1 mL semen was sorted for 45-60 min. Four flow sorters were used, so not more than 4 aliquots could be processed simultaneously. After sorting 3-4 h, semen was pooled and a charge number was assigned to this fraction of the ejaculate (called a freezerun). This procedure was done a maximum of 3 times. The time between collection of the raw ejaculate and freezing of the semen was 7-10 h for freezerun 1, 10-14 h for freezerun 2, and 13-18 h for freezerun 3. From September 2007 to April 2009, a total of 16,523 inseminations with sexed semen from 43 different Holstein bulls were performed for which non-return rates at 56 days (NRR 56) were recorded. There were no significant differences in NRR56 between the 3 freezeruns (t-test) (Table 1). The results shown in this study suggest that raw Holstein semen can be held at room temperature for up to 13-18 h prior to sexing and freezing without an effect on NRR56. This study did not indicate what the maximum holding time prior to sexing and freezing would be before resulting in a fertility decline. Table 1.Number of records and NRR56 for different freezeruns [ABSTRACT FROM AUTHOR]

Two strategies to improve the conception rates in dairy heifers identified in estrus using tail head painting and inseminated with sexed semen were evaluated. In the first trial, the effect of GnRH injection (100 μg of gonadorelin) at estrus on ovarian responses and conception rates (CR) were verified. A total of 413 virgin Jersey heifers 11 to 12 mo of age were used. All heifers were treated with 25 mg of dinoprost tromethamine at the first day of the AI program. All inseminations were performed 12 h after estrus detection using sexed semen (2.1 million sperm) and the estrus was detected based upon removal of tail-head paint. At estrus detection, heifers were randomly assigned to receive GnRH or not (GnRH = 212 and No GnRH = 201). A subset of heifers (GnRH = 70 and No GnRH = 70), was scanned by ultrasound every 12 h after estrus detection until ovulation. The largest follicle diameter at estrus (LF), ovulatory follicular diameter (OF), ovulation rate (OR), interval between beginning of estrus and ovulation (IEO), and the proportion of heifers presenting late ovulation (LO, ovulation occurred with more than 36 h after the beginning of estrus) were recorded. The GnRH did not influence the LF (13.3 ± 0.9 v.13.4 ± 0.2 mm; P= 0.87), the OF (13.4 ± 0.3 v.13.9 ± 0.2 mm; P= 0.17), or OR [98.6% (69/70) v.97.1% (68/70); P= 0.57]. However, GnRH treatment tended to shorten the IEO (29.2 ± 1.1 h v.32.0 ± 1.3 h; P= 0.07) and reduced the occurrence of LO (2.9% (2/69) v.16.2% (11/68); P= 0.02). However, GnRH injection did not influence the CR [GnRH = 47.2% (100/212) v.No GnRH = 51.7% (104/201); P= 0.38]. The second trial evaluated the CRin dairy heifers after the use of single or double insemination dose. A total of 576 Jersey heifers receiving first, second, or third insemination services were used. The heifers were assigned according to sire utilized into one of the three groups (Single 12 h, Double 12 h, or Double 12/24 h). In Single 12 h (n= 193) or Double 12 h (n= 193) groups, heifers received one or two insemination doses (2.1 million sperm) of sexed semen 12 h after estrus detection, respectively. The Double 12/24 h (n= 190) group heifers received two doses 12 h apart from each other (first dose at 12 h and second dose at 24 h after estrus detection). Differences were found between the number of AI services (P= 0.004) on the CR [First = 55.3%a (115/208); Second = 46.1%a (94/204); Third = 34.8% (57/165); P= 0.004]. However, there was no effect of treatment on the CR [Single 12 h = 45.1% (87/193); Double 12 h = 44.0% (85/193); Double 12/24 h = 49.5% (94/190); P= 0.51]. In conclusion, the GnRH injection altered the timing of ovulation, but failed to increase the CR. Furthermore, double insemination doses of sexed semen did not influence the CR in dairy heifers. Dalhart Jersey Ranch-TX, USA and Sexing Technologies-SP, Brazil. [ABSTRACT FROM AUTHOR]

Several studies using sex-sorted sperm by flow cytometry have shown that its fertility is reduced. Therefore, this study evaluated structural and functional characteristics of sperm sexed by flow cytometry. In addition, in vitroembryo production (IVP) and development was assessed when frozen-thawed unsorted and sex-sorted sperm from 4 Nellore bulls. Each ejaculate was separated into three fractions: non-sexed (NS), sexed for X-sperm (SX), and sexed for Y-sperm (SY). After thawing, each sample was analyzed for sperm motility by computer-assisted semen analysis (CASA, Berkeley, CA), sperm head agglutination, sperm morphology, membrane integrity by propidium iodide (PI) and 6-carboxy-fluorescein diacetate (CFDA) staining, acrosome integrity by peanut agglutinin (PNA), capacitation by chlortetracycline (CTC), and chromatin integrity by acridine orange staining. Then, the samples were placed in 45 : 90% (NS90) or 45 : 60% (NS60, SX, and SY) Percoll™ gradients. After Percoll™ centrifugation, sperm pellets were analyzed or used for IVP. All analyses were replicated independently three times. For IVP, 2,271 in vitromatured oocytes were used. To assess fertilization rate, presumptive zygotes were fixed and stained with lacmoid at 18 h post-insemination (hpi). Cleavage was evaluated at Day 2 (48 hpi) and blastocyst development at Days 6, 7, 8, and 9 of culture. Data were analyzed using generalized linear models. No differences (P> 0.05) were observed between SX and SY groups for e sperm variables evaluated either before or after Percoll™. However, non-sexed sperm had higher sperm motility, greater percentage of sperm with intact membranes, and greater percentage of live sperm with intact acrosomes than sexed sperm (P< 0.05). An effect of Percoll™ was observed in the non-sexed samples, with those submitted to 45 : 90% gradient having higher motility, greater percentage of cells with intact membrane, and lower recovery rate than those submitted to a 45 : 60% gradient. No differences among groups were observed for fertilization rate, being 74.0 ± 5.7, 63.2 ± 5.1, 67.2 ± 5.7, and 55.4 ± 5.9% for NS90, NS60, SX, and SY, respectively. Group NS90 showed a greater cleavage rate than did the SY group, while groups NS60 and SX had similar rates to the others. Blastocyst development rates on Day 6 to Day 9 were greater for group NS90. For example, on Day 8 the blastocyst rate was 34.9 ± 3.6, 22.2 ± 3.2, 18.1 ± 3.3, and 14.8 ± 2.9% forNS90, NS60, SX, and SY groups, respectively. All groups showed similar embryonic developmental stages on Day 6 to Day 9. Although sex-sorting affected sperm characteristics, it did not cause a decrease with in vitrofertility. However, differences in blastocyst rates between groups NS60 and NS90 indicated that the sperm selection protocol affected embryo production. Financial support: Embrapa Genetic Resources and Biotechnology. [ABSTRACT FROM AUTHOR]

The high commercial demand on production of genetically valuable female cattle was emphasized after use of sex-sorted semen in commercial in vitroembryo production (IVEP) programs and a reduction of interest in the use of embryo biopsy followed by PCR. Brazil is a leader in IVEP of bovine embryos, and frozen-thawed, sex-sorted samples become a new option for embryo sexing. There are only a few reports in the literature using this new technique for IVEP. Most reports have involved the use of frozen-thawed then sorted for use with AI in Europe. In the present study, frozen semen from three Nelore bulls was used and the blastocyst production rate was evaluated at Day 7 of IVP and compared to previous data from the same bulls and a batch of non-sorted semen. Cumulus oocyte complexes obtained from high genetic merit donors by ovum pickup were matured (TCM-199, supplemented with FCS, LH, FSH, estradiol, pyruvate, and antibiotics) for 24 h and fertilized (Fert-TALP supplemented with BSA, phenylalanine, and heparin) for 18-22 h (Day 0) in vitro.Frozen semen straws were thawed at 35°C for 30 s in a water bath and then selected by centrifugation at 800 gon discontinuous Percoll™ gradients (45 :90%). Samples of frozen-thawed sex-sorted semen were processed from straws from bulls that in some cases may already be dead. Frequently, 3 to 5 straws were thawed, flow-sorted, and then centrifuged to provide sufficient sperm to fertilize 100 oocytes. On Day 1, presumptive zygotes were transferred to culture media (SOFaa supplemented with BSA and FCS) and on Day 7 blastocyst production rate was evaluated. The present results were commercially satisfactory because the frozen-thawed sex-sorted semen from all three bulls performed comparably and, in one case, better than non-sorted semen from the same bulls. Therefore, its use is extremely attractive in commercial bovine IVEP systems and is a more efficient system than embryo sexing. Table 1.Embryo production rates at 7 days post insemination [ABSTRACT FROM AUTHOR]

Births of predetermined-sex calves illustrates the importance of sperm sexing technology for in vitroproduction of bovine embryos (Johnson, 2000). Flow cytometry techniques for sperm sexing are usually performed on fresh semen. However, many bulls presenting high genetic value died or became unproductive before development of sperm sexing technologies. The technique of fresh semen sexing and semen reverse sorting method is based on separation of sperm according to differences in DNA content (X or Y). The objective of this study is to examine the blastocysts and pregnancy rates of bovine embryos produced by fertilization with sperm sexed with the post-thawing procedure, called reverse sorting. Oocytes were obtained by ovum pick-up from Dairy Gir, Nelore, and Guzera cows. The COC were transported to the laboratory and matured at 38.5°C and 5% of CO2in air, TCM-199 medium with FCS (10% vol/vol), FSH (1.0 μg mL-1), hCG (50 μg mL-1), estradiol (1.0 μg mL-1), sodium pyruvate (0.20 mM), and amicacin (83.4 μg mL-1). IVF was performed 24 to 26 h after the onset of maturation. Doses of commercialized semen (3 to 5) from the specified breeds were sent to Goyaike Brazil Agropecuária Ltda. The semen sexing process was performed by flow cytometry and semen was returned to the IVF laboratory at 18°C. In the Tecgene laboratory, semen was centrifugated using Percoll gradient and evaluated. After 18 h, the presumptive zygotes were transferred to IVC in SOF medium at 38.5°C and 5% CO2in air. The cleavage rate was evaluated 48 h after IVF and embryos were transferred at Day 7. A total of 5213 viable oocytes were obtained from 266 donors, fertilized with 7 different reverse sorted semen samples (1 Dairy Gir, 2 Nelore, and 4 Guzera) resulting in 1333 embryos. From these, 1084 transferred embryos resulted in 260 pregnancies to this time, with 226 females and 34 males. Each bull was evaluated separately according to cleavage rate, production of blastocysts, pregnancy rate, and percentage of females, respectively. Bull 1 presented 100%, 38%, 25%, and 90%; bull 2: 98%, 38%, 21%, and 90%; bull 3: 84%, 23%, 21%, and 84%; bull 4: 87%, 15%, 29%, and 86%; bull 5: 92%, 53%, 70%, and 71%; bull 6: 78%, 15%, 34%, and 71%, and bull 7: 55%, 27%, 50%, and 78%. The average of in vitroblastocysts (26%), the pregnancy rate (24%), and the percentage of females (87%) are similar to rates obtained by commercial fresh semen sexed by Goyaike Brazil Agropecuária Ltda. In conclusion, semen reverse sorting is an alternative method for selecting sex of in vitroproduction of bovine embryos using thawed semen collected from extinct or unproductive bulls. [ABSTRACT FROM AUTHOR]

Noninvasive techniques have been successful in distinguishing gender in dimorphic amphibian models (Szymanski DC et al.2006 Zoo Biol. 25, 35-46). The aim of this study was to assess gender-specific fecal hormone metabolite concentrations in 2 dimorphic and 2 monomorphic, critically endangered species of amphibians to assist in ex situbreeding efforts. Fecal samples from 2 dimorphic anurans, the Puerto Rican Crested Toad (Bufo lemur) and the Wyoming Toad (Bufo baxteri) and 2 monomorphic species, the Mountain Chicken Frog (Leptodactylus fallax) and the Dusky Gopher Frog (Lithobates sevosa) were measured by enzyme immunoassay for sex-specific hormone differences (Student''s t-test; P< 0.05) among 6 variables (concentrations of estradiol (E2), progesterone (P), and testosterone (T), and ratios of P:E2, T:E2, and P:T). Values are presented as means ± SEM. Mountain Chicken Frog samples were obtained from individuals of known sex. Dusky Gopher Frog samples were from confirmed females but males were presumed based on subtle physical characteristics. The E2 concentrations (ng g-1) were significantly higher in females than males (P< 0.05) in all species except for the Dusky Gopher Frog. However, males in all 4 species, including the presumptive Dusky Gopher Frog males, exhibited significantly higher P:E2 and T:E2 ratios than females. Additionally, Wyoming Toad and Mountain Chicken Frog females had significantly higher P:T ratios than males (0.19 ± 0.05 v. 0.06 ± 0.00 and 0.34 ± 0.03 v. 0.20 ± 0.02, respectively). Similar to the American Toad (Szymanski et al.2006), presumptive Dusky Gopher Frog males measured significantly higher T concentrations compared with females (376.4 ± 118.5 ng g-1v. 174.0 ± 23.1 ngg-1, respectively). Although species-specific differences are apparent, fecal hormone measurements can be used to identify sex in monomorphic species and offer a potential tool for the captive breeding of endangered anurans. Table 1. [ABSTRACT FROM AUTHOR]