Polymerase Chain Reaction (PCR) is a Useful and Low-Cost Tool for Molecular Sexing Psittaciformes under Human Care: An Example of a Collaborative Approach in Mexico.

Sex determination in monomorphic birds is a precondition for captive breeding programs and management and conservation strategies for threatened species. Most species of the order Psittaciformes often present complications since these birds lack external sexual phenotypic traits, making it impossible to differentiate males and females. In the present study, we used molecular techniques to determine the sex of 31 individuals belonging to nine species of the order Psittaciformes kept under human care at the Akumal Monkey Sanctuary & Rescued Animals in Quintana Roo, Mexico. This is a useful and low-cost methodology based on the analysis of the conserved region of the CHD1 gene, which was amplified by PCR with two sets of primers: P8/P2 and 2550F/2718 R. All individuals were successfully sexed with the first set of primers, while only 28 out of 31 samples (90%) could be amplified with the second set. Out of the 31 individuals analyzed, fifteen are female, and seventeen are male. This information represents a handy tool for adequately managing birds under human care, resulting in their reproduction and eventual reintegration into their natural habitat.

Evaluation of the efficiency of TaqMan duplex real-time PCR assay for non-invasive pre-natal assessment of foetal sex in equine.

Accurate diagnosis of foetal sex in pregnant mare is helpful for many breeders, both for private or commercial purposes. In this study, in order to pre-natal foetal sexing in equine, we used TaqMan duplex real-time PCR to detect the specific regions of SRY and TSPY genes on extracted cell-free foetal DNA from maternal blood. Peripheral blood samples from 50 pregnant Arabian mares with singleton foetuses were collected. Cell-free foetal DNA was extracted from maternal plasma, and duplex real-time PCR assays were performed with TaqMan probes and primers. Amplification of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was used as control of DNA extraction procedure. From the 50 sampled mares, 28 cases had female and 22 mares had male foetuses. The final results for 46 samples were conclusive, and from them, 43 cases were predicted correctly. Sensitivity, specificity and accuracy of the test were 90.48%, 96% and 93.48%, respectively. In conclusion, a TaqMan duplex real-time PCR was set up to pre-natal detection of foetal sex in equine. The method was fast and decreased the false-positive and false-negative results. The technique can be used as a routine procedure in farms by collecting only a blood sample.

Application of embryo biopsy and sex determination via polymerase chain reaction in a commercial equine embryo transfer program in Argentina.

Embryo biopsy for fetal sexing has clinical application, but few reports are available of its use within an active embryo transfer program. We evaluated results on biopsy of 459 embryos over one breeding season. There were no significant differences in pregnancy rate between biopsied and non-biopsied embryos (72% vs 73%) or for biopsied embryos recovered at the centre (73%) compared with those shipped overnight (72%). However, the pregnancy rate decreased significantly in shipped embryos biopsied ≥20h after collection. Overall, 86% of biopsies provided a sex diagnosis. The likelihood of a positive genomic (g) DNA result was significantly higher for biopsies from large blastocysts (96%) than from smaller embryos (70-85%). In total, 38% of biopsies were positive for Y chromosome DNA (Y-DNA) and were diagnosed as male. Subsequently, 95% of Y-DNA-positive embryos were confirmed as male and 78% of Y-DNA-negative embryos were confirmed as female. The accuracy of prediction of female (Y-DNA negative) was significantly higher when the biopsy sample was probed for Y-DNA only compared with probing for both gDNA and Y-DNA. We estimate that by transferring only Y-DNA-negative embryos, 3% of potential female pregnancies may have been lost, and production of male pregnancies was reduced by 72%.

Comparative evaluation of the cost and efficiency of four types of sexing methods for the production of dairy female calves.

This study was conducted to evaluate and compare the economic benefits of different embryo sexing methods, based on the cost per female dairy calf produced. Female calves were produced from four kinds of female embryos: (1) those collected from superstimulated donors at 7-8 days after artificial insemination (AI) with X-sorted semen; (2) those sex-determined by loop-mediated isothermal amplification assay of a biopsy sample of embryos collected from superstimulated donors after AI with conventional unsorted semen; (3) those obtained by invitro embryo production (IVEP), using X-sorted semen and in vitro-matured oocytes collected from donors by ovum pick-up (OPU); and (4) those obtained by IVEP, using X-sorted semen and oocytes collected by OPU after dominant follicle ablation and follicle growth stimulation of the donors. The respective productivities of female calves per technical service and the total production cost per female calf of each sexing method were compared. The production cost per female calf (66,537 JPY), as calculated from the number of female calves per service (1.30), pregnancy rate of transfer (42.9%), rate of female calves obtained (92.9%), and total cost of the method (56,643 JPY plus embryo transfer fee), was less for IVEP with X-sorted semen and follicular growth-stimulated (FGS) oocytes than for the other groups (P < 0.05). The results demonstrate that embryo production with X-sorted semen and FGS oocytes provides a more efficient method for producing female calves than the other embryo sexing methods.

Bioeconomics of sexed semen utilization in a high-producing Holstein-Friesian dairy herd.

A bioeconomic, stochastic spreadsheet model, that included calculation of the net present value of the additional value of all future descendants resulting from increased selection intensity, was developed to study the profitability of using sexed semen in a high input-high output dairy herd. Three management strategies were modeled: (1) only heifers inseminated with sex-sorted semen and cows inseminated with unsorted semen; (2) both heifers and cows inseminated with sex-sorted semen; and (3) a reference scenario, in which all breeding females were inseminated with unsorted semen. A Monte Carlo simulation (@risk software, Palisade Corp., Ithaca, NY) was run to study the sensitivity of net profit and sexed semen advantage to key input parameters. Most input parameters were given truncated normal distributions, whereas the maximum numbers of inseminations in heifers and cows were given discrete distribution functions. The calculated intensity of selection accounted for the different numbers of dairy females born for each of the 100,000 iterations. Using sexed semen (X-sorted, female) was shown to be profitable, with insemination of both heifers and cows being most profitable. The returns on assets were higher when only heifers were inseminated with sexed semen (8.54% ± 2.94; ±SD) or all females were inseminated with sexed semen (8.85% ± 2.93) than when all females were inseminated with unsexed semen (8.38% ± 2.95). The range in net profit was most sensitive to the assumed distributions of milk protein price (€/kg), milk fat price (€/kg), cow pregnancy rate, fertilizer price (€/t), and concentrate price (€/t) when unsorted semen was used. When only heifers or both heifers and cows were inseminated with sex-sorted semen, the range in net profit was most sensitive to the same distributions, with fertilizer price and cow pregnancy rate in reverse order of sensitivity. However, the range in sex-sorted semen advantage (in net profit) when only heifers were inseminated with sex-sorted semen was most sensitive to the assumed distributions of cow pregnancy rate, sex-sorted semen pregnancy rate as a percent of unsorted semen rates, standard deviation of index, additional cost of sex-sorted semen (€/dose), dairy bull calf price (€/head), and dairy heifer calf price (€/head). When both heifers and cows were inseminated, the order of importance of the last 2 inputs was reversed. This study highlights the relatively high effect of pregnancy rate and the genetic value of dairy bulls in determining the level of financial advantage from using sex-sorted semen in a dairy herd.

RAD SNP markers as a tool for conservation of dolphinfish Coryphaena hippurus in the Mediterranean Sea: Identification of subtle genetic structure and assessment of populations sex-ratios.

Dolphinfish is an important fish species for both commercial and sport fishing, but so far limited information is available on genetic variability and pattern of differentiation of dolphinfish populations in the Mediterranean basin. Recently developed techniques allow genome-wide identification of genetic markers for better understanding of population structure in species with limited genome information. Using restriction-site associated DNA analysis we successfully genotyped 140 individuals of dolphinfish from eight locations in the Mediterranean Sea at 3324 SNP loci. We identified 311 sex-related loci that were used to assess sex-ratio in dolphinfish populations. In addition, we identified a weak signature of genetic differentiation of the population closer to Gibraltar Strait in comparison to other Mediterranean populations, which might be related to introgression of individuals from Atlantic. No further genetic differentiation could be detected in the other populations sampled, as expected considering the known highly mobility of the species. The results obtained improve our knowledge of the species and can help managing dolphinfish stock in the future.

Bovine embryo sex determination by multiplex loop-mediated isothermal amplification.

In cattle, the ability to determine the sex of embryos before embryo transfer is beneficial for increasing the number of animals with the desired sex. This study therefore developed a new modification of loop-mediated isothermal amplification in a multiplex format (multiplex LAMP) for highly efficient bovine embryo sexing. Two chromosomal regions, one specific for males (Y chromosome, S4 region) and the other common to both males and females (1.715 satellite DNA), were amplified in the same reaction tube. Each target was amplified by specifically designed inner primers, outer primers, and loop primers, where one of the S4 loop primers was labeled with the fluorescent dye 6-carboxyl-X-rhodamine (emitting a red color), whereas both satellite loop primers were labeled with the fluorescent dye fluorescein isothiocyanate (emitting a green color). After amplification at 63 °C for 1 hour, the amplified products were precipitated by a small volume of cationic polymer predispensed inside the reaction tube cap. Green precipitate indicated the presence of only control DNA without the Y chromosome, whereas orange precipitate indicated the presence of both target DNAs, enabling interpretation as female and male, respectively. Accuracy of the multiplex LAMP assay was evaluated using 46 bovine embryos with known sex (25 male and 21 female) generated by somatic cell nuclear transfer and confirmed by multiplex polymerase chain reaction. The multiplex LAMP showed 100% accuracy in identifying the actual sex of the embryos and provides a fast, simple, and cost-effective tool for bovine embryo sexing.

Ultrasonographic fetometry and prenatal fetal sex assessment in camels (Camelus dromedarius).

The aims of this study were to determine the developmental patterns of some fetal parts to achieve a high accuracy level in the assessment of gestational age and to assess the feasibility and accuracy of ultrasonic prenatal fetal sex assessment in camels. Serial ultrasonographic examinations were carried out on seven pregnant dromedary camels. A total of 329 ultrasonographic examinations were conducted between the second and the 54th weeks of pregnancy. Intrauterine fluid accumulation was detected between the second and third weeks of pregnancy. The embryo proper was noticed between the third and fourth weeks. Organization of the embryo was first observed between the sixth and seventh weeks. Ossification was first detected between the seventh and ninth weeks. The accessibility during the total gestational period was 35/329 (10.6%) for crown-rump length, 35/329 (10.6%) for biparietal diameter, 42/329 (12.8%) for abdominal diameter, 42/329 (12.8%) for ruminal length, and 126/329 (38.3%) for eyeball diameter. A high correlation was found between gestational age and each of the studied parameters (P < 0.0001). The highest correlation was found with the crown-rump length and the biparietal diameter during the first trimester and with the eyeball diameter during the third trimester of pregnancy. The overall accuracy of the ultrasonic prenatal fetal sex assessment was 91.7%. The best window was found during the 11th week of pregnancy. It was concluded that sonographic fetometry can be useful for the evaluation of fetal development, the estimation of gestational age, and the prediction of prenatal fetal sex in camels.

Ultrasonic assessment during pregnancy in goats - a review.

Obstetric ultrasonography is the most common diagnostic approach used in veterinary reproduction because it is a simple, reliable and non-invasive imaging technique. With advances in ultrasonography of small ruminants, assessment of pregnancy in goats is challenging for accurately managing reproduction. This article presents an up-to-date review of the use of ultrasonography in pregnancy for the diagnosis and evaluation of intrauterine foetal growth in goats.

Quality assessment of bovine cryopreserved sperm after sexing by flow cytometry and their use in in vitro embryo production.

The objective was to evaluate the structural and functional quality of bull sperm after sexing by flow cytometry. Frozen non-sexed (NS), sexed for X (SX) and sexed for Y (SY) sperm from four bulls was used. Frozen-thawed sperm was analyzed for motility, sperm head agglutination, morphology, capacitation, and integrity of the plasma membrane, acrosome, and chromatin. After Percoll centrifugation (45:60% gradients), the pellet was used for sperm analysis or IVF. Data were analyzed using generalized linear models (P < 0.05) and were reported as least squares means ± standard error (SEM). Based on sperm evaluations, NS sperm had better (P < 0.05) quality than sexed sperm, including higher motility and greater percentages of cells with an intact membrane and acrosome (58.0 ± 3.0, 58.2 ± 3.0, and 60.9 ± 3.3) than SX (29.6 ± 1.3, 36.0 ± 2.9, and 37.1 ± 3.3), and SY (26.2 ± 2.1, 36.4 ± 2.9, and 37.5 ± 3.3). There were no differences (P > 0.05) among groups for fertilization and cleavage rates. Similarly, blastocyst rate on Day 8 (Day 0 = day of insemination) did not differ among groups (22.2 ± 3.2, 18.1 ± 3.3, and 14.8 ± 2.9 for NS, SX, and SY, respectively). Regarding embryo development kinetics, all groups had similar developmental stages from Days 6 to 9. Although the sex-sorting procedure affected sperm characteristics, it did not significantly affect fertilization or embryo development.

Ultrasonographic assessment of early pregnancy diagnosis, fetometry and sex determination in goats.

This study was conducted to use B-mode trans-rectal (TR) and trans-abdominal (TA) ultrasonography to determine early pregnancy and fetometry as crown-rump-length (CRL) and bi-parietal-diameter (BPD). A total of 110 does aged between 8 and 36 months were used. The accuracy for detecting early pregnancy (fetal fluids and heartbeats) and fetal number (single or twins) was measured. The relationship between gestation age and CRL or BPD was determined from day 40 to 109 of gestation. The accuracy of fetal sexing was determined by differentiation of genital tubercle (GT) at different stages of gestation (from day 40 to 109 of gestation). The examination revealed 95.5% of examined does were pregnant, with accuracy 100% in detecting pregnancy for positive cases. The fetal number was 45.7% and 54.3% for single and twins+triplets, respectively. The TR probe enabled the reliable and earlier recognition of fetal fluid and heartbeats (indicating pregnancy) than TA probe. For maximum reliability, the TR observation of heartbeats is recommended as conclusive evidence of the presence of a live fetus. The TA convex probe was used from day 40 to 89 for measuring CRL and from day 40 to 109 for measuring the BPD. Gestation equations were: CRL=0.464x-17.767 and BPD=0.055x-1.431 (x=gestational age in days). The relation between gestational age and CRL or BPD was highly (p<0.0001) significant. The fetal sexing was found in 100, 83.3 and 64.3% of single pregnancies and in 85.7, 80 and 52.3% of twins+triplets pregnancies during 40-60 days, 61-70 days and 71-109 days of gestation, respectively. The accuracy of sex identification among the 3 groups was not significantly (p>0.05) higher in single than twins+triplets pregnancies. However, identification of GT in male fetus was possible from day 40 onward. From a total of 105 scanned does, 80 (76.25) were sexed. In conclusion, B-mode real-time ultrasonography is recommended as a reliable mean for early detection of gestation as early as 19-27 days after mating, for CRL or BPD measuring as well as fetal sex determination from day 40 of gestation onwards under field conditions.

Accuracy of early fetal sex determination by ultrasonic assessment in goats.

The objective of this study was to determine the accuracy of early fetal sex determination by ultrasonic assessment of the relative location of the genital tubercle (GT) in goats at different stages of pregnancy as well as by the identification of fetal external genitalia. Pregnant animals were divided into three experimental groups (EI: n=21, EII: n=28, EIII: n=33). In EI, fetuses (n=27) were transrectally monitored daily from days 40 to 60 of pregnancy with a linear transducer (6.0 and 8.0MHz). In EII, fetuses (n=40) were examined once between days 45 and 70 of pregnancy by transrectal ultrasonography. In EIII fetuses (n=52) between days 100 and 120 of pregnancy, were submitted to a single transabdominal ultrasonography using a convex transducer (5.0 and 7.5MHz). Regardless of fetal sex diagnosis, 15/15 (EI), 13/16 (EII) and 9/14 (EIII) of single pregnancies and 10/12 (EI), 20/24 (EII) and 21/38 (EIII) of twin pregnancies were correctly identified. The accuracy of sex identification among EI (92.6%), EII (82.5%) and EIII (57.7%) was not statistically different (P>0.05). Identification of the GT in male fetuses was possible from day 45 onward. Changes in the GT position were not observed between days 53 and 60 of pregnancy. Accuracy of fetal sexing under field conditions is high in goats when ultrasound imaging is properly timed during pregnancy and when it is performed with proper equipment by experienced operators.

The current status and future of commercial embryo transfer in cattle.

A commercially viable cattle embryo transfer (ET) industry was established in North America during the early 1970s, approximately 80 years after the first successful embryo transfer was reported in a mammal. Initially, techniques for recovering and transferring cattle embryos were exclusively surgical. However, by the late 1970s, most embryos were recovered and transferred nonsurgically. Successful cryopreservation of embryos was widespread by the early 1980s, followed by the introduction of embryo splitting, in vitro procedures, direct transfer of frozen embryos and sexing of embryos. The wide spread adoption of ethylene glycol as a cryoprotectant has simplified the thaw-transfer procedures for frozen embryos. The number of embryos recovered annually has not grown appreciably over the last 10 years in North America and Europe; however, there has been significant growth of commercial ET in South America. Within North America, ET activity has been relatively constant in Holstein cattle, whereas there has been a large ET increase in the Angus breed and a concomitant ET decrease in some other beef breeds. Although a number of new technologies have been adopted within the ET industry in the last decade, the basic procedure of superovulation of donor cattle has undergone little improvement over the last 20 years. The export-import of frozen cattle embryos has become a well-established industry, governed by specific health regulations. The international movement of embryos is subject to sudden and dramatic disturbances, as exemplified by the 2001 outbreak of foot and mouth disease in Great Britain. It is probable that there will be an increased influence of animal rights issues on the ET industry in the future. Several companies in North America are currently commercially producing cloned cattle. The sexing of bovine semen with the use of flow cytometry is extremely accurate and moderate pregnancy rates in heifers have been achieved in field trials, but sexed semen currently is available in only a few countries and on an extremely limited basis. As of yet, all programs involving the production of transgenic cattle are experimental in nature.

Sexing mammalian sperm--intertwining of commerce, technology, and biology.

Sperm from many mammalian species can be sexed by flow cytometry/cell sorting at about 90% accuracy without damaging them unduly. However, because sperm are evaluated one at a time, in series, the number of sexed sperm produced per unit time is limited. Furthermore, the equipment required currently is expensive, in the order of 300,000 US dollars per machine. Despite these limitations, commercialization of this technology has begun with bovine semen, in part by inseminating cattle with relatively low number of sperms. No other approach to sexing sperm in any practical way is likely to be available within the next few years. The constraints for commercial application of sexed sperm in cattle can be somewhat lowered fertility, the high costs of equipment and skilled personnel, and costs of intellectual property such as licensing fees and royalty payments. Most economic analyses indicate that farmers can afford to pay 10-20 US dollars more per dose of sexed sperm than unsexed sperm if costs must be recouped by selling milk or meat. When the product is breeding stock or for certain niche applications, a higher price for sexed semen may be justified. Sexed sperm will be used more broadly in cattle only when improved production and/or efficiency can compensate for the extra costs of purchasing sexed sperm.

Comparative efficiency and accuracy of surgical and cytogenetic sexing in psittacines.

Otoscopic surgical sexing and chromosome analysis through lymphocyte culture were undertaken in 22 psittacine birds of eight species for the purpose of establishing their sex. Comparison of the techniques involved considering success rate, quality of determination, cost, efficiency, and risk. Surgical endoscopy appears to be preferable to chromosome analysis in all categories except risk.