**4.1. AI following estrus detection**

**1.** Frozen conventional semen (40 x 106

**3.** Frozen sexed semen (1 x 106

**4.** Cooled sexed semen (5 x 106

Breed Category

*Timed Artificial insemination*

*Artificial insemination with estrus detection*

Cows and

Cows and

Dairy Heifers

Dairy

Dairy

**2.** Frozen conventional semen at low concentration (1 x 106

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

sperm/dose);

sperm/dose).

non sex-sorted sperm (conventional = 67% and low concentration = 49%)

Conventional (%) (n/n)

Dairy Cows 27.1 (44/162) 13.0 (21/161) 48.0

Beef Heifers 67.6 (96/142) 53.7 (130/242) 79.4

Beef Heifers 67.0 (85/126) 52.6 (129/245) 78.5

56.0 (30082/53718)

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

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

> > Sexed (%) (n/n)

Beef Cows 54.2 (232/428) 45.4 (193/425) 83.7 Sá Filho et al. [31] (Exp

Beef Cows 54.7 (134/245) 45.9 (113/246) 83.9 Sá Filho et al. [31] (Exp

Beef Cows 51.8 (100/193) 41.8 (82/196) 80.7 Sales et al. [8] Beef Cows 55.3 (105/190) 40.9 (79/193) 74.0 Sales et al. [8]

Dairy Heifers 60.0 (1375/2292) 38.0 (881/2319) 63.3 DeJarnette et al. [24]

Dairy Cows 46.0 (69/149) 21.0 (33/157) 45.6 Andersson et al. [33]

Heifers 37.7 (160/426) 22.9 (51/223) 60.7 Mellado et al. [32]

Heifers 37.4 (34/91) 28.8 (38/132) 77.0 Bodmer et al. [29]

45.0 (17893/39763)

sperm/dose) deposited at uterus corpus;

sperm/dose);

Proportion (%)

Reference

1)

2)

Souza et al. 2006 unpublished data

Seidel and Schenk [17] (Exp.1)

Seidel and Schenk [17] (Exp.2)

80.3 DeJarnette et al. [30]

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 was performed 16.2 h before ovulation.

In a review by Seidel et al. [26], crossbred beef heifers inseminated with sex-sorted sperm, present conception rate about 40%, lower than AI with non sex-sorted sperm (75%).

removal. Timed artificial insemination use to occur 48 to 60 hours after P4/progestin source

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

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

47

A possibility to improve the use of sex-sorted sperm is controlling the ovulation time varia‐ tion through the use of synchronization techniques; thus, increasing the efficiency of AI pro‐ grams using sexed semen. For instance, in beef and dairy cows, P4 and E2 based

The P4-based synchronization protocol is a well-established protocol to synchronize the ovu‐ lation. Despite the satisfactory predictability of the moment of ovulation provided by the P4 plus estradiol-based estrus synchronization protocol (averaging 66 to 72 h after P4 device re‐ moval), the timing of ovulation is influenced by the diameter of the follicle at the time of the ov‐ ulatory stimulus treatment [62]. Neves [62] evaluated the time of ovulation in a large number of suckled *Bos indicus* cows (n = 312) and observed a significant effect of the diameter of the ov‐ ulatory follicle at the moment of synchronized ovulation (average of 71.8 ± 7.7 h after P4 de‐ vice removal). The author reported that cows experiencing premature ovulation (i.e., ovulation occurring from 48 to 59 h after P4 device removal) presented a larger ovulatory folli‐ cle (14.0 ± 2.2 mm) than cows with delayed ovulation (11.4 ± 2.2 mm; 73 to 96 h after P4 device removal) and that cows that ovulated at the expected time of ovulation (60 to 72 h after P4 de‐

synchronization induce ovulation around 70-72h after the P4 device removal [59-61].

vice removal) showed ovulatory follicles of intermediate diameter (13.6 ± 2.1 mm).

Once the sex-sorted sperm present lower viability on the reproductive tract than conven‐ tional semen [6, 14], our research group has evaluated the delay on AI using of sex-sorted sperm in heifers. A study [8] breeding 420 cyclic Jersey heifers at either 54 or 60 h after P4 device removal, using either sex-sorted (2.1 x 106 sperm/straw) or non-sorted sperm (20 x 106 sperm/straw) from three sires (2 x 2 factorial design). There was an interaction (P = 0.06) be‐ tween time of AI and type of semen on pregnancy per AI (P/AI, at 30 to 42 d after TAI); it was greater when sex-sorted sperm (P < 0.01) was used at 60 h (31.4%; 32/102) than at 54 h (16.2%; 17/105). In contrast, altering the timing of AI did not affect conception results with non-sorted sperm (54 h = 50.5%; 51/101 versus 60 h = 51.8%; 58/112; P = 0.95). There was an effect of sire (P < 0.01) on P/AI, but no interaction between sire and time of AI (P = 0.88).

Based on previous results, Sales et al. [8] evaluated the ideal period to perform the TAI with sex-sorted sperm in a P4-based protocol of synchronization of ovulation. Suckled *Bos indicus* cows (n = 339) were randomly assigned to receive TAI with sex-sorted sperm at 36, 48, or 60 h after P4 device removal. Ultrasonographic examinations were performed twice daily in all cows to confirm ovulation. On average, ovulation occurred 71.8 ± 7.8 h after P4 removal, and greater P/AI was achieved when insemination was performed closer to ovulation. The P/AI was greatest (37.9%, 36/95) for TAI performed between 0 and 12 h before ovulation, whereas P/AI was significantly less for TAI performed between 12.1 and 24 h (19.4%, 21/108) or > 24 h (5.8%, 5/87) before ovulation (P = 0.001) as shown on Table 4. In the Table 5, it is presented a summary of studies when AI with sex-sorted sperm is performed at different times after

protocol of synchronization of ovulation.

withdrawal [54-58].

Sá Filho et al. [7] have been evaluated the different times to perform the AI. Thereby, 638 Jersey heifers have been inseminated after estrus detection according those times (12 a 16h; 16 a 20h; 20 a 24h e 24 a 30h) and the estrus has been detected using radio telemetry (Heat Watch®). The P/AI of heifers inseminated from 12 to 16 h after the onset of estrus (40/106 = 37.7%) was less (P = 0.03) than those inseminated from 16.1 to 20 h (85/164 = 51.8%), and 20.1 to 24 h (130/234 = 55.6%). However, the P/AI for heifers inseminated from 24.1 to 30 h (61/134 = 45.5%) did not differ from that of any other interval.

Pharmacological manipulation is expected to increase the reproductive performance even in management with estrus detection. Exogenous GnRH given at the onset of estrus [45, 46] or concurrent with AI [47, 48] have improved fertility, but the effects have not been consistent [45-48]. Therefore, reproductive strategies to enhance fertility with exogenous hormones, optimizing estrus detection, or improving the timing of AI relative to estrus detection, could enhance the use of sexed semen in dairy cattle breeding. Following this idea, our group aimed to develop strategies to improve P/AI (35 to 42 d after AI) in virgin Jersey heifers bred by AI of sex-sorted sperm after being detected in estrus [7]. Nevertheless, giving 100 μg of GnRH at first detection of estrus, with AI 12 h later, did not affect P/AI in females with es‐ trus detected by tail-head chalk [GnRH=47.2% (100/212) vs. No GnRH=51.7% (104/201); P = 0.38] or by radio telemetry [HeatWatch® ; GnRH=53.1% (137/258) vs. No GnRH=48.6% (122/251); P = 0.43]. In the referred study, GnRH treatments were done 7.4 h after the onset of estrus, identified by HeatWatch® system, due to the management schedule (i.e. twice dai‐ ly 07:00 or 19:00). Previous studies demonstrated that the onset of estrus, the peak of the 17β-estradiol in plasma, and the release of the ovulatory LH surge occurred at approximate‐ ly the same moment [49, 50]. However, treatment with GnRH following a spontaneous LH surge resulted in a surge of LH of shorter duration and decreased magnitude compared to an ovulatory LH surge [51]. Additionally, treatment with GnRH at AI tended to decrease subsequent progesterone concentrations in synchronized beef heifers [48]. Therefore, the positive effect of GnRH treatment at estrus appeared to be most beneficial in females with decreased fertility, or when the treatment was performed close to the onset of estrus (i.e., close to the spontaneous LH surge).
