Semen Sex Research Articles

Tingkat keberhasilan Inseminasi Buatan (IB) menggunakan semen beku hasil sexing pada Sapi Persilangan Ongole

The purpose of artificial insemination using sexing semen was to obtain the calf according to the expected sex. This study aimed to determine the success of artificial insemination in non-sexing and sexing of frozen semen. The materials used in this research were 77 non-sexing artificial insemination acceptor cows and 77 sexing artificial insemination acceptor cows with double dose treatment. The method used in this study was experimental field experiment with the research treatment: P0 = frozen non-sexing semen straw and P1 = frozen semen of spermatozoa Y by sexing method using Percoll Density Gradient Centrifugation (SGDP) produced by Balai Besar Inseminasi Buatan (BBIB) Singosari. The variables of this study were Non Return Rate (NRR), Service Per Conception (S/C) and Conception Rate (CR). The data obtained were analyzed descriptively. The results of the study in each non-sexing and sexing artificial insemination treatment were: the percentage of NRR 1 were 90.91% and 77.92%, followed by NRR 2 with 88.31% and 76.62%, the pregnancy percentage were 84.42% and 75.33%, and the CR values were 79.22% and 57.14%. The conclusion of this study was artificial insemination using frozen non-sexing semen had a higher success rate compared to artificial insemination using frozen sexing semen.

Influence of breed and technological factors on the fertility of repair heifers sorted by sex semen of Holstein bulls

Influence of breed and technological factors on the fertility of repair heifers sorted by sex semen of Holstein bulls

Consequences of manipulating gametes and embryos of ruminant species

During the past 12 years, ruminants have provided a focus for some significant advances in mammalian reproductive biotechnologies. Lambs were the first offspring generated after nuclear transfer of fetal or adult cells to enucleated oocytes, and many calves of pre-determined gender are today the result of commercialized semen sexing. In 1990, the birth of one calf provided living proof that even 'dead' spermatozoa can be paternal, whereas, more recently, a short-lived gaur calf and viable mouflon lamb represented a novel option for conservation of endangered species. As well as highlights, hazards have emerged, resulting in setbacks or developmental anomalies, such as those associated with the large offspring syndrome which encompasses a range of adverse fetal, placental and post-natal phenomena expressed in ruminants. In this review, the developmental and other consequences of applying manipulative procedures, such as assisted fertilization, semen sexing, cloning and gene transfer, to gametes and embryos from bovine, ovine and caprine species are considered. Although assisted fertilization techniques can overcome mammalian infertility, they also usurp natural gamete selection safeguards, but not always with impunity. In the case of manipulations such as cloning, and to a lesser extent gene transfer, it is evident that nuclear-cytoplasmic interactions and nuclear-mitochondrial DNA interdependences are at least partially damaged or destroyed with a view to reconstruction. Therefore, among surviving zygotes and embryos it is inevitable that the legacy is frequently one of altered genetic, epigenetic or cellular programmes and processes.

Combining extended lactation with sexed semen in a dairy cattle herd: Effect on genetic and total economic return

ABSTRACT Extended lactation has shown economic benefits in dairy herds, but this strategy may affect the genetic lag between the production cows at herd level and the semen bulls unfavorably. We hypothesize, that a breeding strategy using sexed semen (SS) will reduce this effect and that SS and extended lactation has synergetic effects on the total economic return. A base scenario reflected an average Danish Holstein herd with 13 months calving interval (CInt) (BASE). Additionally, six scenarios with extended lactations: two with only first parity cows managed for a CInt of 15 (FIRST15) and 17 months (FIRST17), two with only cows in second parity or higher managed for a CInt of 15 (OLDER15) and 17 months (OLDER17), and two with all cows managed for a CInt of 15 (ALL15) and 17 months (ALL17). These were combined with four breeding schemes illustrating different uses of SS within each of the scenarios: SS in 50% genetically best heifers (SS50), 75% genetically best heifers (SS75), all heifers (SS100) and 70% genetically best heifers and 30% first parity cows (SS7030). Beef semen was used when necessary to equalize the number of replacement heifers within extended lactation scenarios. The scenarios were simulated using two stochastic simulation models for estimation of operational returns (SimHerd) and for genetic returns (ADAM). The genetic returns support that extended lactation has an unfavorable effect on genetic lag in SS0, because the generation interval among breeding females increases when extended lactation is introduced. Compared to BASE/SS0 most combinations of SS and extended lactation, with the exception of ALL17 combinations increased genetic returns as a result of decreased generation interval. Across extended lactation scenarios, reductions in genetic return relative to BASE was between €1 (FIRST15) and €12 (ALL17) per cow year within each scenario of SS. With increased use of SS, the genetic returns increased from €4 (SS50) to €12 (SS7030) per cow year, compared to SS0 within each scenario of extended lactation. In total economic return, the use of SS had effects between +€5 and +€29 per cow year due to increased use of beef semen, thus more sales of slaughter calves. Except for OLDER15 and OLDER17, the extended lactation scenarios within each scenario of SS increased total economic returns as well. In conclusion, the genetic lag caused by extended lactation can be reduced using SS, although there was no synergetic effect between SS and extended lactation on the total economic return.

Effect of two pre-freezing methods on quality of sexed semen in Ettawa Grade goat

The aim of this study was to determine the influence of pre-freezing different procedures to the quality of Ettawa Grade goat frozen semen-sexing. The research material was semen sexing consisted of 2 layers those were top layer and bottom layer. The quality of frozen semen sexing was observed by comparing the pre-freezing technique (factory standard operating procedure: FSOP) according to the Artificial Insemination Center operating procedure (pre-freezing by placing the straw of semen for about 4 cm above liquid nitrogen for 9 minutes) with the modification procedure (MP), pre-freezing by placing the straw of semen 16 cm above liquid nitrogen for 9 minutes and then it was lowered to 4 cm above liquid nitrogen for 9 minutes during the pre-freezing phase. The parameters observed were motility, progressive motility, hyperactivation, and sperm linearity. Data were analyzed using Student's t-test.The results of this study indicated that the quality of sexed-semen in the standard operating procedures of frozen semen compared to the treatment of modifications to the top and bottom layers were motility at the top layer (46.06 ± 7.52% vs 55.6 ± 7.78%) and bottom layer (36.82 ± 6.49% vs. 41.47 ± 6.57%); progressive top layer (16.34 ± 4.27 vs. 32.83 ± 5.9%) and bottom layer 15.97 ± 2.72% vs. 19.79 ± 3.97%); hyperactivity in the top layer (0.81 ± 0.6% vs 4.09 ± 1.98%) and the bottom layer (0.71 ± 0.68% vs. 1.50 ± 1.05%); linearity consisted of linear and non-linear, the top layer (12.19 ± 2.94 vs. 20.52 ± 3.97%) and bottom layer (12.32 ± 2.63 vs 14.70 ± 2.6); while non-linear in top layer (0.14 ± 0.2 vs 0.68 ± 0.85%) and bottom layer (0.4 ± 0.13% vs 0.34 ± 0.4%). The conclusions in this study indicated that the quality of the frozen sexed-semen that has processed by pre-freezing modification technique was better than the frozen sexed-semen obtained from the Artificial Insemination Center Standard Operating procedure.

Conception rate hasil inseminasi buatan menggunakan semen sexing beku pada Sapi Persilangan Ongole

<p>Artificial Insemination (AI) is a technique that is widely used by farmers to improve the genetic quality of livestock. This method can be increased in value by using sexing semen that can regulate and produce the sex of the offspring as expected. The purpose of this study was to determine the success of AI by using frozen sexing semen on Ongole Crossbred cows in Malang Regency. The materials used were 98 Ongole crossbred cows in traditional farm of Malang Regency especially in Pakis, Tumpang, Jabung and Singosari Districts. The method used was a field experiment with two treatments: 49 cows were inseminated by using frozen sexing semen (P1) and 49 cows were inseminated by using non sexing semen (P0). The variables observed were the Non-Return Rate (NRR) value observedin 3 estrous cycle after AI and CR value based on pregnancy observation at 8 months of pregnancy. Data were analyzed descriptively and continued with a one-proportion z-test. The datashowed that the CR value on P1; P0 were 55.10% and 65, 31% respectively.furthemore, the NRR value on P1 from NRR<sub>1</sub> to NRR<sub>3 </sub>were 87,76%, 73,47% and 67,35%, while on P0 95,92%, 83,67% and 75,51% respectively.It can be conclude thatCR and NRR value of sexing semenwas lower than non sexing semen, however sexing semen can be applied for certain purposes such as producing offspring as expected</p>

Keberhasilan inseminasi buatan menggunakan semen sexing beku pada Sapi Persilangan Ongole

<p>The Artificial Insemination (AI) results in Beef cattle are expected to be male calf because they can increaseweight gain rapidly, so in this study AI was carried out by using sexing semen with Percoll density gradient centrifugation method containing Y sperm.This study was conducted to evaluate the success rate of AI using frozen sexing semen on Ongole Cross Breed. The study method used wasexperimental, comparing AI frozen sexing semen and frozen semen in 54 Ongole Cross Breed. The study variableswere Non Return Rate<sub>1</sub> (NRR<sub>1</sub>), Non Return Rate<sub>2</sub> (NRR<sub>2</sub>) and Conception Rate (CR).The study showed that the success rate of based on the value of NRR<sub>1</sub> and NRR<sub>2</sub>AI using frozen sexing semen on Ongole Cross Breed is lower than un sexed frozen semen with the NRR<sub>1</sub> value is 73,91%; 93,54% while the value of NRR<sub>2</sub> was 69,56%; 83,87%. Based on CR value of frozen sexing semen higher than un sexed frozen semen showed CR valuewas 43,47%; 35,48%.</p>

English

The aim of this review paper was to investigate the use of biotechnology in the improvement of animal reproduction in different Asian Countries. Biotechnologies have contributed immensely to increasing livestock reproductively, particularly in developed countries, and can help to alleviate poverty and hunger, reduce the threats of diseases and ensure environmental sustainability in developing countries. A wide range of biotechnologies are available and have already been used in different Asian countries in the main animal science disciplines, that is animal reproduction, genetics and breeding; animal nutrition and production; and animal health. In animal reproduction, genetics and breeding, artificial insemination (AI) has perhaps been the most widely applied animal biotechnology, particularly in combination with cryopreservation, allowing significant genetic improvement for productivity as well as the global dissemination of selected male germ plasm. Complementary technologies such as semen sexing can improve the efficiency of AI. Embryo transfer provides the same opportunities for females, albeit on a much smaller scale and at a much greater price. Molecular DNA markers can also be used for genetic improvement through marker assisted selection (MAS) as well as to characterize and conserve animal genetic resources. Specific options that should assist Asian countries make informed decisions regarding the adoption of appropriate biotechnologies in the livestock sector in the future. Key words: Animal biotechnology, Asian countries, Reproduction.

Sperm Sexing of Dairy Cattle:Economics, Animal Welfare and Technological Challenges

There is a growing need for sexed semen of cattle in India. Farmers prefer female calves of cows or buffalos than male calves as they can produce milk in the future which is economically more beneficial. Very few bulls are required for breeding in herds and they are no more required in agricultural fields due to mechanization of cultivation and transportation. Therefore these extra bulls/oxen are often sold at low prices to the meat industry. This is an animal welfare concern and can also cause communal tensions due to differences in belief systems of religious sects. At present there is no indigenous technology for producing sexed semen although artificial insemination with nonsexed semen is a well-established method of breeding among cattle farmers. The imported sexed semen is not easily available and also not affordable for most Indian farmers. There is a need for the development of cost effective indigenous sperm sexing techniques as an alternative to fluorescence-activated cell sorting. Recent developments in microfluidics and BEMS (bio electromechanical systems) may hold the key to the development of a portable device for semen sexing with minimal tampering of the sperm structure.

Reproductive Biotechnologies in Indian Cattle

In recent years there have been immense improvements in application of reproductive biotechnologies in ca ttle and include artificial insemination, embryo transfer, ovum pick up and in vitro fertilization, semen sexing, cloning, transgenics and xenotransplantation. The purpose of these technologies had been improvement of the genetics of animals or producing a nimals of benefit to human beings. The application of these technologies in indigenous dairy cows of India had been slow. This manuscript describes the status of some of the reproductive biotechnologies as applied to Indian breeds of cows.

Impact of applying sex sorted semen on the selection proportion of the sire of dams selection pathway in a nucleus program.

ObjectiveIn a nucleus breeding scheme, the sire of dam’s pathway plays an important role in producing genetic improvement. Selection proportion is the key parameter for predicting selection intensity, through truncating the normal distribution. Semen sexing using flow cytometry reduces the number of vials of sperm that can be obtained from a proved bull. In addition, a lower fertility of this kind of sperm is expected because of the lower sperm dosage in sex sorted semen. Both of these factors could affect the selection proportion in the sire of dam’s pathway (p SD).MethodsIn the current study, through a deterministic simulation, effect of utilizing sex sorted semen on selection (p SD) was investigated in three different strategies including 1: continuous use of sex sorted semen in heifers (CS), 2: the use of sex sorted semen for the first two (S2) and 3: the first (S1) inseminations followed by conventional semen.ResultsResults indicated that the use of sex sorted semen has a negative impact on the sire of dams (SD) pathway due to increase in selection proportion. Consequently selection intensity was decreased by 10.24 to 20.57, 6.38 to 8.87 and 3.76 to 6.25 percent in the CS, S2, and S1 strategies, respectively.ConclusionConsidering the low effect of sexed semen on genetic improvement in dam pathways, it is necessary to consider the joint effect of using sex sorted semen on the sire and dams pathway to estimate about the real effect of sexed semen on genetic improvement in a nucleus breeding scheme.

Reproductive performance of sexed versus conventional semen in Holstein heifers in various semiarid regions of Iran

For comparing reproductive performance of sexed versus conventional semen a total of 3573 heifer insemination records collected from five herds in four provinces of Iran were investigated. The studied provinces were classified into three regions: hot semiarid (Tehran and Alborz provinces), temperate semiarid (Khorasan Razavi province) and cold semiarid (Zanjan province). Various parameters including the conception rate, number of services per conception, calf sex ratio, calf birth weight, gestation length, calving ease score, abortion and stillbirth as well as twining rate were investigated. The logistic regression method was deployed for the analysis of categorical variables while the GLM procedure was applied for the analysis of continuous variables. The average conception rate of sex sorted and conventional semen was 48.3 and 63.8%, respectively. The utilisation of sex sorted semen resulted in 91.1% female calves. The conception rate, number of services per conception, gestation length, calf birth weight and the calf sex ratio were among the reproductive variables that were significantly influenced by the semen sexing. A greater number of services per conception and calving ease score were observed in hot and temperate semiarid areas. These climatic regions also provided a lower incidence of female calf births compared to the cold semiarid regions. Our results highly reaffirm the previous findings on the reproductive performance of sex sorted semen. Yet, climatic and management practices of a herd within a special climate have to be considered when deciding for utilisation of sex sorted semen in a dairy farm.

Current status, scope and constraints of sexed semen - An Indian perspective

The aim of sexed semen is to produce a calf of a specific sex. The use of sexed semen increases the rate of genetic gain not only from the daughter-dam path but also through production of superior male from elite cows for future breeding. Gender selection using sexed semen from genetically elite bulls is imperative to meet the projected demand of 191.3 million tones of milk by 2020 in the country. The demand of sex semen in dairy cattle is also increasing in order to dispose the large number of unproductive males, to ensure required number of progenies per bull under progeny testing programme and to reduce the replacement cost on heifers. In India, this technique is gradually been adopted by many states like Punjab, Haryana, West Bengal, Kerala etc. However, high cost and lower fertility limits its wider use across the country. Furthermore, there is need to standardize the lower dosage of spermatozoa, site of deposition for AI with good conception rate in Indian conditions. Optimal use of sexing technology also requires excellent and careful animal management (nutrition, disease control, estrus detection, semen handling, and insemination technique) for obtaining higher efficiency. More research needs to be carried out to make it feasible in order to extend this technique in our country.

Application of reproductive technologies to improve dairy cattle genomic selection

Genomic selection implies assessing the breeding value of an animal based on the DNA markers uniformly covering the whole genome. This review summarizes the information on the modern trends in dairy cattle genomic selection and the use of reproductive technologies allowing for the increase of breeding efficiency. The main trends in the advance of genomic selection are the improvement of the accuracy of breeding value estimations by pooling the reference populations; use of female genotyping in breeding programs; imputation of genotypes based on lower marker density SNP arrays; and prediction of animal genotypes from the genotyped relatives. Genomic selection can be even more profitable in combination with the state-of-the-art reproductive biotechnologies, such as semen sexing, multiple ovulation and embryo transfer, ovum pick-up followed by in vitro fertilization, embryo genotyping, and cloning of the best breeders. In dairy cattle genomic selection programs, biotechnological manipulations with gametes and embryos allow for the improvement of many factors determining breeding efficiency, namely, selection intensity, accurate breeding value assessment, and the generation interval. Successful embryo genotyping methods for numerous markers after biopsy at the morula or blastocyst stage are based on the whole-genome amplification of the embryo’s DNA. Eventually, these achievements will provide the grounds for new approaches to reducing the generation interval, selecting elite cows, reducing the inbreeding rate, etc.

Application of reproductive technologies to the improvement of dairy cattle genomic selection

Genomic selection is a direction of breeding in which the value of an animal is predicted from DNA markers evenly covering the entire genome. This review summarizes information on modern trends in the genomic selection of dairy cattle and on application of reproductive technologies to the improvement of breeding process. The main trends in the development of genomic selection include improvement of the accuracy of breeding value estimations by combination of reference populations; use genotyping of cows in breeding programs; imputation of genotypes for absent SNPs with low marker density microarrays, and prediction of animal genotypes from the genotypes of relatives. Genomic selection can be even more profitable in combination with up-to-date reproductive biotechnologies: semen sexing, multiple ovulation and embryo transfer, ovum pick-up followed by in vitro fertilization, embryo genotyping, cloning of best breeders, etc. In programs of dairy cattle genomic selection, biotechnological procedures with gametes and embryos allow improvement of a variety of parameters determining breeding efficacy: selection intensity, accurate breeding value assessment, and generation interval. Successful methods for embryo genotyping for numerous markers after biopsy at the morula or blastocyst stage are based on whole genome amplification of embryo DNA. Eventually, these achievements will provide grounds for new approaches to the reduction of generation interval, selection of elite cows, reduction of inbreeding rate, etc.

Resveratrol as a protective agent of goat sperm submitted to sex sorting in discontinuous Percoll gradient - preliminary results

Background Sexing sperm is an important biotechnical that enables improve animal production, with achievement of maximum animal breeding. However, all sex semen methodologies can promote irreparable damages to sperm cell, which compromise their fertility [1]. Among the main causes of injures to the sperm are the reactive oxygen species, which have their production intensified during semen manipulation [2]. Therefore, antioxidants therapies can be an important alternative to protect the sperm during laboratory manipulation [3]. Thus, as resveratrol is a powerful antioxidant with an important role on sperm protection [3], the aim of this study was to evaluate the protective effect of resveratrol on goat sperm submitted to sex sorting in discontinuous Percoll gradient.

Sperm cryopreservation in wild animals

Extinction of a species represents the loss of a resource evolved through eons of mutations and natural selection. Reproductive technologies, including artificial insemination, embryo transfer, in vitro fertilization, gamete/embryo micromanipulation, semen sexing, and genome resource banking (GRB) have all been developed with the aim of solving existing problems and preserving genetic material for conservation purposes. Although protocols from domestic or non-threatened related species have been extrapolated to nondomestic and endangered species, usually these reproductive technologies are species-specific and inefficient in many nondomestic species because of insufficient knowledge on their basic reproduction biology and the need for species-specific customization. Since spermatozoa are usually more accessible and come in large numbers compared to oocytes and embryos, they are considered the primary cell type preserved in most emerging GRBs. For this purpose, semen from endangered species is currently cryopreserved to avail long-term storage. Due to the intractability of most exotic species, semen collection without chemical restraint is limited to only a handful of species and individuals. Viable epididymal spermatozoa can be obtained from dead or castrated animals, but this resource is limited. Electroejaculation, artificial vagina, abdominal massage, and/or transrectal, ultrasound-guided, massage of the accessory sex glands of living animals are viable alternative methods of semen collection. The ultimate goal is to adapt and optimize collection and cryopreservation protocols for each species, making it feasible, among other things, to collect gametes in the wild and introduce them into captive or isolated populations to increase genetic diversity. Recent advances in these fields have allowed the establishment of GRBs for many threatened species.

Variation among Beef Bulls in the Ratio of X- to Y-Chromosome Bearing Spermatozoa

A study was conducted to evaluate variation in the ratio of X- to Y-bearing sperm of individual ejaculates and to determine any relationship between skewed sex ratio and either routine morphological evaluation or computer assisted sperm analysis (CASA). Semen was collected from bulls weekly for 6 consecutive weeks, sperm DNA was recovered and quantitative, and real-time PCR was used to determine the ratio of X- to Y-bearing sperm in each ejaculate. The overall mean of X-bearing sperm within ejaculates was 54.7% over the 6 weeks of semen collections. The percentages of X-bearing sperm were similar (P > 0.5) across all collections. Between bulls, there were differences (P < 0.05) in the mean percentage of X-bearing sperm. No significant correlations were found between CASA parameters and percentage of X-bearing sperm across bulls, so analysis was done within each bull. Different combinations of CASA and/or morphological parameters were found to correlate with the percentage of X-bearing sperm but the prediction equations were specific for individual bulls and unlikely to be of use across bulls. These results confirm that the ratio of X- to Y-bearing sperm may be skewed in some ejaculates of bull semen. Some sperm parameters measured by CASA or routine morphological evaluation were associated with semen sex ratio.

Improving of pig production by intrauterine artificial insemination and other biotechnological solutions

Thanks to constantly increasing human population number during the last few decades, the need for pork meat on market increased dramatically. That requirement made imperative for improvement of swine production efficiency. To achieve this goal, it is necessary to implement new biotechnological procedures and production protocols. The use of artificial insemination (AI) in intensive swine production increased notably during the last 30 years and there is a constant need for improvement of AI technologies. New insemination methods (insemination with frozen-thawed semen, different insemination techniques) as well as modern equipment for analysis (CASA microscope, flow cytometry) and sperms' triage (semen sexing), contributed to obtaining much better production results in modern pig industry. The aim of our research was to evaluate sows' reproductive performances after the usage of intrauterine (IU) insemination with reduced semen volume doses and reduced sperm number, in comparison on classical, intracervical artificial insemination. The experiment included a total of 200 sows from 3 pig farms, which were divided in two groups. Test group (n=100) was inseminated applying intra-uterine (post-cervical) insemination technique with special balooncatheters, while control group (n=100) was inseminated on the usual manner, using intracervical (IC) technique. Insemination in both groups was done after oestrus detection (standing reflex) in the presence of mature boar. For IU artificial insemination had been used chilled semen doses with 1,5 billion of spermatozoain total volume of 50 ml, while for IC insemination of control group had been used standard semen dose of 100 ml and 3 billion spermatozoa. In both groups, insemination had been done once. Insemination with catheter for IU artificial insemination was successful in 89 sows. From the total number of successfully inseminated sows, 84 sows farrowed (94.38 %), while farrowing rate in control group was 78%. Based on results from this experiment, we determined statistical significant (p<0,01) higher number of liveborn piglets after intrauterine insemination (11,00 ± 1,57) with regard to intracervical insemination (10,14 ± 1,10). Applying of intrauterine (postcervical) insemination technique increases a number of spermatozoa that get into oviduct, on site of fertilization. That raises farrowing rate, litter size and number of live born piglets in inseminated sows. In addition, the use of IU catheters decreases semen backflow which allows usage of semen doses of less volume with reduced number of sperm cells. Insemination with this type of IU catheters can't be done if sows aren't in optimal oestrus period. Insemination of double more sows with the same volume of quality semen, better using of genetic material from excellent boars, better assessment of optimal insemination time especially during warm period of the year, are reason good enough for applying IU techniques on pig.

Introducing and validation of SYBR Green Real-Time PCR method to determinate sex ratio in bovine semen

Flow cytometry is a widely used application for validating the accuracy of sperm sexing. However, this method is relatively expensive and requires considerable technical support. An alternative method employing simpler technology at low cost could be suitable for the evaluation of bovine semen in laboratories with low budgets. We used a SYBR Green Real-Time PCR assay to determinate sex ratio in bovine semen. The PLP and SRY genes were amplified to isolate the specific fragments of X- and Y-chromosome sequences, respectively. Two certified standard curves were obtained using two plasmids containing PLP and SRY amplicons. Our results show no significant difference in semen sex ratio in unsorted semen (54.7±0.52% X and 47.6±0.60% Y). However, significant difference was observed in X/Y-sorted semen (93.3±0.08% X and 91.4±0.06% Y-sperm), as compared to the expected ratio in unsorted semen or the post-sorting reanalysis data. The evolution of X-chromosome bearing sperm content in unsorted samples showed an average of 52.6 for ejaculates and 51.8 for the commercial semen. In order to confirm our results, the accuracy, repeatability and reproducibility of the method were tested resulting in 98.2% accuracy, repeatability of CV=5.59% and reproducibility of CV=5.40%. Thus, this method is demonstrated to be a reliable and inexpensive way to test sexual chromosome content in semen samples.