**3. Sperm concentration for sex-sorted sperm in reproductive programs**

Commercially, the established spermatozoa number by dose of sexed semen is 2.1 x 106 cells/dose. This amount is much lower than that from conventional semen (~20 x 106 cells/ dose). To achieve this proportion many studies have been done to specify the best straw concentration considering commercial aspects. Holstein heifers and cows have same concep‐ tion rate when inseminated with sex-sorted sperm in a dose of 2.1 or 3.5 × 106 sperm/dose; however, not only in heifers but also in cows, there is an increasing on conception when AI is performed with conventionally processed sperm (15 x 106 sperm/dose) as presented on Table 1.


**Table 1.** Different superscript letters (a, b) in row indicates statistical difference (P < 0.01).Adapted from Dejarnette et al. [23].Conception rates Holstein heifers and cows after artificial insemination with 2.1 or 3.5 × 106 sex-sorted sperm or 15 × 106 non sex-sorted sperm.

Dejarnette et al. [24] compared the effects of sperm dosage (2.1 vs. 10 × 106 sperm/dose) and sex-sorting (conventional vs. sexed) on conception rates of Holstein heifers (n = 9,172) and observed difference among groups as follows on Table 2. In a field trial study with three dif‐ ferent breeds (Holstein, Jersey and Danish Red) Borchersen and Peacock [25] observed a re‐ duction of 12% for Holstein, 7% for Jersey, and 5% for Danish Red in conception rate using sexed semen.


**Table 2.** Different superscript letters (a, b, c and d) indicates statistical difference (P < 0.01).Adapted from Dejarnette et al. [24].Conception rates of Holstein heifers according semen type (sexed vs. conventional) and sperm dosage combinations (2.1 vs. 10 × 106 sperm/dose).

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

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‐

**3. Sperm concentration for sex-sorted sperm in reproductive programs**

Commercially, the established spermatozoa number by dose of sexed semen is 2.1 x 106 cells/dose. This amount is much lower than that from conventional semen (~20 x 106 cells/ dose). To achieve this proportion many studies have been done to specify the best straw concentration considering commercial aspects. Holstein heifers and cows have same concep‐ tion rate when inseminated with sex-sorted sperm in a dose of 2.1 or 3.5 × 106 sperm/dose; however, not only in heifers but also in cows, there is an increasing on conception when AI

**Sex-sorted sperm (dose) Non sex-sorted sperm (dose)**

(2,864/6,268) 60.7b (3,805/6,268)

(1,388/5,466) 31,5b (1,722/5,466)

**Sperm dosage** 2.1 x 106 10 x 106

(1,003/2,279)

(881/2,319) 44c

2.1 x 106 3.5 x 106 15 × 106

**Table 1.** Different superscript letters (a, b) in row indicates statistical difference (P < 0.01).Adapted from Dejarnette et al. [23].Conception rates Holstein heifers and cows after artificial insemination with 2.1 or 3.5 × 106 sex-sorted sperm

Dejarnette et al. [24] compared the effects of sperm dosage (2.1 vs. 10 × 106 sperm/dose) and sex-sorting (conventional vs. sexed) on conception rates of Holstein heifers (n = 9,172) and observed difference among groups as follows on Table 2. In a field trial study with three dif‐ ferent breeds (Holstein, Jersey and Danish Red) Borchersen and Peacock [25] observed a re‐ duction of 12% for Holstein, 7% for Jersey, and 5% for Danish Red in conception rate using

Non sex-sorted sperm (%) 55b (1,255/2,282) 60d (1,375/2,292)

**Table 2.** Different superscript letters (a, b, c and d) indicates statistical difference (P < 0.01).Adapted from Dejarnette et al. [24].Conception rates of Holstein heifers according semen type (sexed vs. conventional) and sperm dosage

sperm/dose) as presented on

men grew and developed normally both pre- and postnatally.

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

is performed with conventionally processed sperm (15 x 106

(2,752/6,268) 45.7a

(1,257/5,466) 25,4a

Sex-sorted sperm (%) 38a

Table 1.

Heifers (%) 43.9a

Cows (%) 23.0a

or 15 × 106 non sex-sorted sperm.

combinations (2.1 vs. 10 × 106 sperm/dose).

sexed semen.

In a first report to evaluate the fertility of lactating dairy cows under field conditions, fe‐ males where inseminated with same low concentration (2 x 106 ) of sex-sorted or non sexsorted 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) form the conception rate obtained by conventional semen [27].

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 single insemination dose (2.1 x 106 sperm) 12 h after estrus detection (n=193), a double dose at 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 (First, 115/208 = 55.3%a ; Second, 94/204 = 46.1%a ; and Third, 57/165 = 34.8%b ; P = 0.004). Similar 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 insemination (12 hour interval) have not changed the conception rate.

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 following treatments:


Semen for the Treatment 1 was deposited into the uterus corpus. Semen for the other three treatments was shared where each half was deposited into each uterine horn. Pregnancy was lower for the sex-sorted sperm treatments (frozen = 23% and cooled = 25%) than for the non sex-sorted sperm (conventional = 67% and low concentration = 49%)

**Pregnancy rate based on type of semen used in AI**

**Table 3.** Pregnancy rate of females inseminated with conventional or sexed semen and the pregnancy proportion

In brief, the fertility of cows and heifers is influenced when the dose of the sex-sorted sperm

increasing the insemination dose would make this commercially unviable. Certainly duo to the low sorting rate per hour provided by flow cytometry method. Nowadays, sexing com‐ panies just offer sexed semen in a dose of 2.1 x 106 sperm/straw, and studies have been done using this dose as pattern'. Also, data suggest a better use of sex-sorted sperm in the first/

The optimal time at which insemination should take place relative to ovulation (IOI) depends primarily on the lifespan of spermatozoa and on the viability of the oocyte in the female geni‐ tal tract [35]. Several experiments [36-38] have demonstrated that 6 h is the minimum time needed for a viable sperm population capable of fertilization to pass through the oviduct. Fur‐ thermore, the number of progressive motile sperm peaked from 8 to 18 h after insemination. In terms of the oocyte, the most desirable period for fertilization appears to be between 6 and 10 h after ovulation [39]. Also, Dransfield et al. [40] and Roelofs et al. [41] demonstrated that the probability that conception will occur decreases when AI is performed near the time of ovula‐ tion (less than 12 or 6 h before ovulation, respectively). According to Roelofs et al. [42], fertiliza‐ tion rate drastically decreases when AI occurs after ovulation. Artificial insemination should occur near the time of ovulation to maximize sperm access to the ovum, but not so late that an aging ovum awaits sperm arrival [43]. The ovulation occurs 28-30 h after the estrus beginning. The optimal AI time was between 24 and 12 h before ovulation for the most desirable rate of fertilization and 16–12 h for greatest percentage of greater quality embryos [89% of recovered embryos; [42]. More precisely, Maatje et al. [44] obtained an optimal pregnancy rate when AI

44.3% (19982/45080)

79.2

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

sperm/AI; [24]]. However, the high cost of

Seidel and Schenk [17] (Exp.4)

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

45

Seidel and Schenk [17] (Exp.5)

Dairy Heifers 60.0 (74/124) 46.7 (114/244) 77.8

Dairy Heifers 62.0 (163/263) 42.1 (225/534) 67.9

55.9% (32753/58549)

**4. Timing for AI using non sex-sorted or sex-sorted sperm**

obtained by sexed semen based on conventional. (sexed/conventional).

is considerably increased [for example to 10 x 106

Overall

**4.1. AI following estrus detection**

was performed 16.2 h before ovulation.

second service.



**Table 3.** Pregnancy rate of females inseminated with conventional or sexed semen and the pregnancy proportion obtained by sexed semen based on conventional. (sexed/conventional).

In brief, the fertility of cows and heifers is influenced when the dose of the sex-sorted sperm is considerably increased [for example to 10 x 106 sperm/AI; [24]]. However, the high cost of increasing the insemination dose would make this commercially unviable. Certainly duo to the low sorting rate per hour provided by flow cytometry method. Nowadays, sexing com‐ panies just offer sexed semen in a dose of 2.1 x 106 sperm/straw, and studies have been done using this dose as pattern'. Also, data suggest a better use of sex-sorted sperm in the first/ second service.
