**6. Use of sex-sorted sperm in superovulation protocol for embryo production**

The use of sex sorted sperm in reproduction program using superovulation to produce fe‐ male calf in dairy farms or male in beef farms have been used progressively. Our research group has studied this aspect in Nelore (*Bos indicus*) cows superovulated and inseminated with sex-sorted sperm. Animals were synchronized at Day 0 with norgestomet ear implant (Crestar®, MSD) with 2 mg of estradiol benzoate i.m. (Sincrodiol®, Ourofino). The follicle su‐ per stimulation was done was induced with 8 decreasing doses of pFSH (12/12 h) beginning on Day 4). On Day 6, was given PGF2α analog (Sincrocio®, Ourofino). The ear implant was removed 36 h after the PGF2α analog administration, with the application of LH (Luteotro‐ pin) 48 h after PGF2α analog. The TAI with sex-sorted (4,2x106 cell/AI) or non sex-sorted sperm (40x106 cell/AI) was performed at 12 and 24 h after LH injection. For TAI, it was used semen from same sire. The experimental design used was crossover to avoid individual var‐ iation among donors. The Table 6 summarizes the experiment described, demonstrating a decreasing on fresh and frozen embryos, fresh and frozen embryo rate and an increasing on the unfertilized embryos when using sex-sorted sperm. The accuracy on the use of the sexed semen to produce the desired sex was 90% with pregnancy diagnosis 60 days after TAI. The conventional semen produced 52.7% of females.

variation in the physic-chemical semen properties of the individual bull. A study [65] to ver‐ ify the fertilizing potential of sex-sorted frozen-thawed bull sperm transported cooled or fro‐ zen to the sorting facility, has shown a bull effect on the pregnancy rate after AI [Bull 1: conventional semen (control) 63.0%; previously frozen (FS) 8.6%; previously cooled (CS)

52.8

Conv. Bull 2

**Figure 2.** Pregnancy rate of Jersey heifers fixed timed artificially inseminated according the bull and type of semen

Accordingly, the individual difference among bull is an important aspect to consider apply‐ ing the sex-sorted sperm use at livestock level, allowing the sire selection for higher per‐ formance after sexing. Also, it is essential to highlight that this sire effect is one of the most

The use of sex sorted sperm in reproduction program using superovulation to produce fe‐ male calf in dairy farms or male in beef farms have been used progressively. Our research group has studied this aspect in Nelore (*Bos indicus*) cows superovulated and inseminated with sex-sorted sperm. Animals were synchronized at Day 0 with norgestomet ear implant (Crestar®, MSD) with 2 mg of estradiol benzoate i.m. (Sincrodiol®, Ourofino). The follicle su‐ per stimulation was done was induced with 8 decreasing doses of pFSH (12/12 h) beginning

34.9

Sexed Bull 2

Conv. Bull 3

61.4

21.9

Sexed Bull 3

10.0%. Bull 2: control 45.5%, FS 0%, CS 4.8%; P = 0.001].

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

39.4

Conv. Bull 1

important obstacle to the use of sexed semen in large scale.

14.7

Sexed Bull 1

used (conventional or sexed). It was verified a bull effect (P = 0.001) and type of semen (P = 0.001).

**6. Use of sex-sorted sperm in superovulation protocol for embryo**

**Pregnancy rate (%)**

0

**production**

10

20

30

40

50

60

70


**Table 6.** Embryo production of superovulated Nelore cows (*Bos indicus*) and inseminated in fixed time with sex-sorted or non sex-sorted sperm.

In a study by our group research [66], we evaluated different intervals for TAI with sex-sort‐ ed sperm after pLH treatment in *Bos indicus* and *Bos taurus* donors. The hypothesis was that increased embryo production would occur when TAI with sex-sorted sperm was performed closer to the time synchronized ovulations occurred. In the first experiment, hormonal su‐ perstimulation of ovarian follicular development in Nelore donors (n = 71) was performed in randomly allocated animals to one of three treatment groups, and they were inseminated at 12 and 24 h after an ovulatory stimulus with pLH treatment was applied, either with sexsorted (4.2 x 106 sperm/insemination; S12/24; n = 17) or non-sorted sperm (20 x 106 sperm/ insemination; NS12/24; n = 18), or they were inseminated at 18 and 30 h using sex-sorted sperm (4.2 x 106 sperm/insemination; S18/30; n = 19). A greater number of transferable em‐ bryos were found when sex-sorted sperm was used to inseminate the animals at 18 and 30 h compared to insemination at 12 and 24 h. However, a greater embryo production was ob‐ tained with non-sorted sperm (results are summarized on Table 7).

Additionally, Soares et al. [66] used the same insemination times and semen types in lactat‐ ing high-production Holstein cows (n = 12). A crossover design was employed in this trial. A lesser embryo production (0.007) was found in Holstein donors that were inseminated us‐ ing sex-sorted sperm at 12 and 24 h compared to non-sorted sperm. However, intermediate results were obtained when the inseminations with sex-sorted sperm were performed at 18 and 30 h (Table 8).

Briefly, it is possible to improve embryo production using sex-sorted sperm in *Bos indicus* and *Bos taurus* superstimulated donors when the inseminations are performed near the same time as time-synchronized ovulations. However, the embryo production for TAI with sex-

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

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

53

**7. Strategies to increase the pregnancy in TAI with sex-sorted sperm**

After determining the best moment to perform the TAI with sex-sorted sperm, some studies were conducted aiming to verify the effect estrus expression and follicle diameter at the mo‐ ment of TAI on the conception rate. Earlier studies have demonstrated that females display‐ ing estrus before TAI have better ovarian responses [67] and with bigger follicle size at the moment of TAI [68] have better conception rate when AI is performed with conventional se‐ men. When using this method of the follicle size on TAI with sex-sorted sperm [31], there is an interaction (P = 0.02) between the type of semen and the size of the dominant follicle [conventional ≥ 8mm = 58.9% (126/214); conventional < 8mm = 49.5% (101/204); sexed semen ≥ 8mm = 56.8% (134/236) and sexed semen < 8mm = 31.2% (59/189)]. In this study, it was verified that the difference between the type of semen used (conventional vs. sexed) on the pregnancy probability at 30 days, decrease according the dominant follicle size increase at

There is an influence of the number of services with sex sorted sperm and the conception rate in heifers. It was observed by Sá filho et al. [7] working with Jersey heifers (n = 573) showed that P/AI was influenced by the number of AI service (First, 115/208 = 55.3%a; Sec‐ ond, 94/204 = 46.1%a; and Third, 57/165 = 34.8%b; P = 0.004). Similar results were achieved in a study by Dejarnette et al. [30] where pregnancy rate reduced in heifers with more AI serv‐ ice (First = 47%, Second = 39%, and Third = 32%). Thus, heifers which use to fail in the first

Vazquez et al. [69] in an interesting review, states that the sex-sorted spermatozoa are 'weakened' by the process, giving them a short functional lifespan. Consequently, new AI strategies are necessary in order to achieve high pregnancy rates with a low dose of sex-sort‐ ed spermatozoa. The deposition of the spermatozoa higher in the reproductive tract, com‐ pared with conventional AI, allows a greater proportion of spermatozoa to survive and colonize the oviduct. Therefore, fewer spermatozoa are necessary to achieve the same proba‐ bility of fertility than with a dose deposited in a lower part of the reproductive tract, espe‐ cially when weak spermatozoa are inseminated. However, in a recent study, Sá Filho et al. [31] compared the conception rate of a total of 200 suckled cows presenting LF ≥ 9 mm at TAI were randomly assigned to receive sex-sorted sperm deposited into the uterine body (n = 100) or into the uterine horn ipsilateral to the recorded LF (n = 100). No effect of deeper artificial insemination on P/AI was found (P = 0.57). Several studies have been performed to evaluate the effect of uterine horn insemination [70-77]. The majority of those previous re‐ sults support the idea that site of semen deposition would play little to no role in P/AI. However, due to the presumably reduced viability of sexed sperm, the insemination closer

AI probably will have their pregnancy rate compromised in the later services.

sorted sperm was still less than the production with non-sorted sperm.

TAI (P = 0.001).


**Table 7.** Rows with different superscripts (a, b, e) indicate P < 0.05. d Percentage of transferable embryos based on the number of ova/embryos recovered. e Percentage of freezable embryos based on the number of ova/embryos recovered.Adapted from Soares et al. [66].Embryo production of Nelore cows (*Bos indicus*) superovulated and inseminated in different times with conventional or sexed semen.


**Table 8.** Rows with different superscripts (a, b, c) indicate P < 0.05. d Percentage of transferable embryos based on the number of ova/embryos recovered. e Percentage of freezable embryos based on the number of ova/embryos recovered.Adapted from Soares et al. [66].Embryo production of Holstein cows (*Bos Taurus*) superovulated and inseminated in different times with conventional or sexed semen.

Briefly, it is possible to improve embryo production using sex-sorted sperm in *Bos indicus* and *Bos taurus* superstimulated donors when the inseminations are performed near the same time as time-synchronized ovulations. However, the embryo production for TAI with sexsorted sperm was still less than the production with non-sorted sperm.
