**2. Basic principles of sexing**

The biotechnology of sex sorting is based on the information that X-sperm has about 4% more genetic material than Y-sperm. In this manner, the flow cytometry associates the laser, differential coloration of the viable and non-viable spermatozoa and hydrodynamic force which direct the sperm at the moment of the reading during the process of X and Y sperm separation. Moreover, there are differences among bovine breeds according to the amount of DNA present in the Y chromosome. According Garner [9], the X-Y sperm nuclei DNA content difference (%) is: 4.22 for Jersey, 4.07 for Angus, 4.01 for Holstein, 3.98 for Hereford and 3.7 for Brahman. Such differences, do not determine the fertility after sexing process. These differences mainly determine the speed and efficiency of the sexed semen production and have to be considered when using flow cytometry.

Recent advances in the form of the tip of the flow cytometry, the positioning of the sperm cells at the moment of the passage through the laser, as well as changes in pressure and the type of staining cells have significantly improved separation process gametes X and Y [9]. The X-Y sperm separation speed is relatively slow with approximately 300,000 to 400,000 cells per minute. In this way, for a higher process efficacy, the semen dose in the straw nor‐ mally used is set to be 2.1 x 106 cells in a 0.25cc straw.

Reproductive programs based on TAI, have been continuously incorporated in routine of the reproductive management on farms. These programs represent a systematic approach to enhance the use of AI in dairy or beef farms, increasing the benefits of this reproduc‐

The ET technique has been used widely around the world once it increases the number of offspring that can be obtained from females with great genetic value [2, 3]. The use of sexsorted sperm could increase the production of a specific calf gender, which would benefit beef and dairy industries worldwide [4]. Likewise, the TET synchronizes the ovulation simi‐ lar to TAI; however, instead to inseminate before ovulation, the recipient cow receive an em‐

Advances in sex sorting of sperm using flow cytometry have enabled its incorporation in‐ to commercial reproductive management. Despite the increased use of sex-sorted sperm, preg‐ nancy per AI (P/AI) is still less than when using non sex-sorted sperm [5]. Regardless of these reduced results, suitable spermatozoa concentration at AI time; longer intervals from the induction of ovulation to the AI (i.e., closer to the expected moment of ovulation); AI into the uterine horn ipsilateral to the expected ovulation; the size of the follicle from which ovulation occurs; occurrence of estrus from progesterone (P4) source removal to the TAI and the identification and use of bulls with proven fertility producing spermatozoa resist‐ ant to the sexing process have increased the likelihood of pregnancy in females inseminat‐ ed with sex-sorted sperm [6-8], thereby optimizing the use of sex-sorted sperm in TAI and

There is a huge interest in sex-sorted sperm around the world. There are many opportuni‐ ties and challenges associated to the use of this semen in farms. The aim of this review is to bring into focus a summary of our current understanding of the use of sex-sorted sperm in TAI and ET programs and some strategies to optimize the use of sex-sorted sperm. Before describing the researches results and opportunities for its use, it is important to understand

The biotechnology of sex sorting is based on the information that X-sperm has about 4% more genetic material than Y-sperm. In this manner, the flow cytometry associates the laser, differential coloration of the viable and non-viable spermatozoa and hydrodynamic force which direct the sperm at the moment of the reading during the process of X and Y sperm separation. Moreover, there are differences among bovine breeds according to the amount of DNA present in the Y chromosome. According Garner [9], the X-Y sperm nuclei DNA content difference (%) is: 4.22 for Jersey, 4.07 for Angus, 4.01 for Holstein, 3.98 for Hereford and 3.7 for Brahman. Such differences, do not determine the fertility after sexing process. These differences mainly determine the speed and efficiency of the sexed semen production

bryo fertilized *in vitro* with sex-sorted sperm seven days after ovulation.

40 Success in Artificial Insemination - Quality of Semen and Diagnostics Employed

how sperm are sorted and the critical points associated to the process.

and have to be considered when using flow cytometry.

tive biotechnology.

ET programs.

**2. Basic principles of sexing**

The process of sorting X and Y-bearing sperm likely results in some damage to the sperm that compromise fertilization [4, 10]. According to Gonsálvez et al. [11], the sorting process produce an interaction of the DNA with fluorophores, laser exposure, spermatozoa separa‐ tion in micro-droplets, acceleration of spermatozoa through geometrically-pressured fluid channels and centrifugation. All of these para-biological spermatozoa-media or mechanical interactions would theoretically have the potential to produce changes in cell structures, in‐ cluding the DNA molecule. When considering cell structures, spermatozoa appear to be par‐ tially capacitated during the flow cytometry process used for sex pre-determination [12]. This total or partial capacitation is induced by the conditions that sperm are subjected dur‐ ing preparation for flow cytometric-sorting and during sorting [13]. Lu and Seidel [12] em‐ phasize that it could be due to the condition that the sperm are pre-incubated with Hoechst 33342 at 34.5 8C for 45 min before sorting. During sorting, sperm are subjected to laser light and various physical forces, such as exiting the sorter at nearly 90 km/h before entering the collecting medium. The process of sorting results in an extremely diluted sample with 800,000 sperm/ml, and subsequently sperm are gently centrifuged to provide a concentrated sample suitable for packaging and cryopreservation.

Thus, this process could also led to a shorter functional sperm life compared to non-sorted sperm [12, 14, 15], which could include pre-capacitation and a reduced number of viable spermatozoa for the insemination [6, 16]. The thermo-resistance test showed that the motili‐ ty decline in sex-sorted sperm was faster compared to non-sorted sperm. Also, there is an effect associated with samples from a specific bull and sex-sorted sperm insemination dose [5] and some samples from certain bulls can tolerate the stress of sorting in a more desirable manner [17].

Although the above information stated, it is important to highlight that AI with sex-sorted sperm does not alter pattern of return to estrus and does not affect the likelihood of heifers to conceive from subsequent AI [18]. Also, Holstein heifers inseminated with sexed semen had similar pregnancy loss from 29 ± 1 to 50 ± 1 d after AI compared with heifers inseminat‐ ed with conventional semen [17], and there is no difference on abortion rate from 2 mo of gestation to parturition. Farmers in general are interested to know if calves produced by sexed semen are different that those from conventional semen. To address this question Tubman et al. [19] analyzed data from 1,169 calves produced from sexed semen and 793 calves from conventional semen. They did not observed difference in gestation length, birth weight, calving ease, calf vigor, weaning weight, abortion rate, and death rates (neonatal and through weaning) among calves produced by sexed or conventional semen. When *in vi‐ tro* models are used to verify the efficiency of sex-sorted sperm to produce embryos, there are inconsistent results concerning the embryo development which mainly depend on the sire used [20-22]. In general, P/AI of females inseminated with sex-sorted is not resultant from increased late embryonic and fetal losses. Therefore, calves produced from sexed se‐ men grew and developed normally both pre- and postnatally.

In a combination of some experiments, Seidel et al. [26] observed that the conception rate of Holstein heifers inseminated with sex-sorted sperm vary from 40% to 68%, and with nonsex-sorted sperm vary from 67% to 82%. Also, Seidel and Schenk [17] observed a lower pregnancy rate when using sex-sorted sperm (31% to 42%) than non sex-sorted sperm (43% to 62%). Although the greater variability on the pregnancy outcomes of cattle inseminated of with sex-sorted sperm by literature, most part of the researches with heifers indicates that conception rate after AI upon estrous detection with sex-sorted sperm is about 70% to 90% (according to the farms handling) from the conception obtained following the use of conven‐ tional semen [27]. In accordance, Sá Filho et al. [28] showed that overall P/AI rates were re‐ duced with sex-sorted sperm compared with non sex-sorted sperm (i.e., 83.8% pregnancy was obtained with the non-sex-sorted sperm). This reduced P/AI could be attributable to several factors including a shorter lifespan in the female reproductive tract, reduced number of sperm per straw, and sperm damage from the staining, identification, and separation processes [5, 6, 14]. The Table 3 summarizes the main studies with sex-sorted sperm, stating

The Use Of Sex-Sorted Sperm For Reproductive Programs In cattle

the pregnancy rate and the proportion of pregnancy sexed/conventional semen.

males where inseminated with same low concentration (2 x 106

form the conception rate obtained by conventional semen [27].

; Second, 94/204 = 46.1%a

insemination (12 hour interval) have not changed the conception rate.

single insemination dose (2.1 x 106

(First, 115/208 = 55.3%a

following treatments:

In a first report to evaluate the fertility of lactating dairy cows under field conditions, fe‐

sorted frozen-thawed sperm [29], it was observed the same pregnancy per AI among females inseminated with sex-sorted (27.6%, n = 105) or non sex-sorted (28.1%, n = 64) sperm. Although the inconsistent results with cows presented by literature, most part of the researches with heifers indicates that conception rate after estrus detection by observation and AI with sex-sorted sperm is about 70% to 90% (it depends on to the farms handling)

Considering that maybe straw concentration would still be low, our research group per‐ formed an experiment inseminating Jersey heifers once or twice [7]. Aimed at, 576 virgin Jer‐ sey heifers were synchronized with two injections of PGF2α apart and had their estrus observed twice daily (based upon removal of tail-head chalk). The AI was performed with a

12 h (n=193), or a double dose involving insemination 12 and 24 h after estrus detection (n=190). It was not observed any effect of treatments on P/AI (87/193 = 45.1%, 85/193 = 44.0%, and 94/190 = 49.5%, respectively; P = 0.51). However, P/AI was influenced by the number of AI service

results have also been described by Dejarnette et al. [30] where, pregnancy rate has been re‐ duced in heifers when the number of AI service has been increased (First service = 47%; Second = 39% ; Third = 32%). Accordingly, in Jersey heifers, the increasing on the spermatozoa num‐ ber, 2.1 to 4.2 million, to be used in insemination after estrus detection and to perform a double

The use of sex-sorted sperm in suckled beef cows in the post-partum have not been much explored scientifically. Most part of the papers use few cows per treatment, making the re‐ sults inconclusive. A study in suckled Angus cows (n = 212), Doyle et al. [34] compared the

sperm) 12 h after estrus detection (n=193), a double dose at

; and Third, 57/165 = 34.8%b

) of sex-sorted or non sex-

http://dx.doi.org/10.5772/52180

43

; P = 0.004). Similar
