**4.2. AI following synchronization of ovulation**

The use of a P4/progestin plus E2 based TAI protocols has been the most commercially used type of fixed time synchronization protocol in South America [52-55]. In females, a common aspect among the estrus synchronization protocols for TAI is the insertion of an intravaginal device containing P4 or an ear implant containing norgestomet plus administration of estra‐ diol benzoate (EB; 2mg i.m.) on Day 0; an injection of prostaglandin (PG) F2α on Day 8 or 9 at the moment of device withdrawal plus 300 to 400 IU of equine chorionic gonadotropin (eCG). Different ovulation inducers with similar efficiency could be used such as estradiol cipionate (EC; 0.5 mg i.m.) at moment or EB (1mg i.m.) 24 h after the P4/progestin implant removal. Timed artificial insemination use to occur 48 to 60 hours after P4/progestin source withdrawal [54-58].

In a review by Seidel et al. [26], crossbred beef heifers inseminated with sex-sorted sperm,

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

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.,

The use of a P4/progestin plus E2 based TAI protocols has been the most commercially used type of fixed time synchronization protocol in South America [52-55]. In females, a common aspect among the estrus synchronization protocols for TAI is the insertion of an intravaginal device containing P4 or an ear implant containing norgestomet plus administration of estra‐ diol benzoate (EB; 2mg i.m.) on Day 0; an injection of prostaglandin (PG) F2α on Day 8 or 9 at the moment of device withdrawal plus 300 to 400 IU of equine chorionic gonadotropin (eCG). Different ovulation inducers with similar efficiency could be used such as estradiol cipionate (EC; 0.5 mg i.m.) at moment or EB (1mg i.m.) 24 h after the P4/progestin implant

present conception rate about 40%, lower than AI with non sex-sorted sperm (75%).

(61/134 = 45.5%) did not differ from that of any other interval.

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

close to the spontaneous LH surge).

**4.2. AI following synchronization of ovulation**

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

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‐ 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.


**5. Bull effect on the efficiency of reproductive programs using sex-sorted**

An important factor to consider in the timing of AI with sex-sorted sperm is the variation in the fertility of individual bulls. Whereas sperm sorting has significantly decreased fertility of certain bulls, sperm sorting does not affect fertility of other bulls [25]. Sales et al. [63] have been evaluated the use of the sexed or conventional semen of 3 different sires to inseminate Jersey heifers after the estrus detection by radio telemetry (Heat Watch®). Wherefore, the conventional semen [64.2% (238/371)] had been a higher conception rate than sexed semen [49.5% (189/382); P = 0.001]. Moreover, there was a bull effect on the conception rate [Bull A = 50.0% (108/216)b; Bull B = 63.4% (211/333)a and Bull C = 53.5% (107/200)b; P = 0.008]. Thus, some bulls can present lower conception rate using sexed or conventional semen for insemi‐ nation (Figure 1). Other studies also have described that conception rates vary in magnitude

**Bull 755 Bull 760 Bull 762**

Bull A Touro A BullB Bul C

**Figure 1.** Conception rate of Jersey heifers inseminated artificially with sexed or conventional semen according three

In another study, Sales et al. [8] synchronized Jersey heifers and used sex-sorted sperm from three different sires to inseminate the females. The conception rate was different among sires used in the experiment, indicating once more the existence of individual discrepancy

The few number of sorting facilities around the world limits the use of high genetic merit bulls because the distance. The capacity to effectively sex-sort and re-freeze previously fro‐ zen-thawed sperm would allow commercial sorting undertaking to offer sex-sorted sperm from any sire currently in the frozen semen market. This capacity is mainly influenced by

different the bull (A, n=216; B, n= 333 e C, n=200). Bull effect (P = 0.008) and semen (P = 0.001).

among bulls producing semen to sex-sorting (Figure 2).

56.1

Sexed semen Conventional semen

70.9

40.4

59.8

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

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

49

**sperm**

for individual sires [5, 25, 64].

0

20

40

**Pregnancy rate (%)**

60

49.5

57.6

80

**Table 4.** a,b Within a column, proportions without a common superscript differed (P \_ 0.05).x OR, odds ratio; CI, confidence interval.y Reference, reference group for adjusted risk ratio, which is the industry standard for the optimal timing of AI with non-sorted sperm.z Inseminations were performed within 0–12 h after ovulation.Adapted from [8]Risk of pregnancy based on the interval between TAI and ovulation in suckled B. indicus cows inseminated with sexsorted sperm.


**Table 5.** Influence of the AI moment in synchronization of ovulation protocols on the pregnancy rate.

Importantly to note, the use of in vitro fertilized (IVF) embryos with sexed semen is expect‐ ed to have its use increased throughout the years associated to TET. The overall percentages of oocytes fertilized with sorted and unsorted frozen bovine sperm appear to be similar us‐ ing current IVF methods [20]. While TAI uses one dose of sexed semen by cow, the TET al‐ lows the optimization of the semen use. Just one sexed semen dose is capable to fertilize about 80 oocytes (equivalent to the aspiration of four females; mean of 20 oocytes per aspi‐ rated cow), resulting in the production of approximately 30 viable embryos. Considering that TET provides 40 – 50% of pregnancy rate, transferring the 30 embryos fertilized with just one sexed semen dose, would result theoretically, in 12 – 15 pregnancies. In the case where for TAI is considered the same conception rate of 40 – 50%, each inseminated sexed semen dose would result in just 0.4 – 0.5 pregnancies.
