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

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

> Sexed 12/24

Conventional 12/24

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

Number of cows 17 18 19 ---- Total ova/embryos 8.0 ± 3.2 7.1 ± 3.3 9.0 ± 3.8 0.14 Transferable embryos (n) 6.8 ± 2.6a 2.4 ± 1.8c 4.5 ± 3.0b < 0.001 Transferable embryos (%)d 86.1 ± 11.9a 37.3 ± 26.7c 48.2 ± 25.9b < 0.001 Freezable embryos (n) 6.0 ± 2.4a 2.0 ± 1.4c 3.7 ± 2.8b < 0.001 Freezable embryos (%)e 76.3 ± 19.2a 31.8 ± 24.5c 38.0 ± 26.5b < 0.001 Degenerate embryos 0.7 ± 0.7 0.9 ± 1.6 1.6 ± 2.1 0.05 Unfertilized oocytes 0.5 ± 0.7a 3.7 ± 3.6b 2.9 ± 2.6b < 0.001

**Table 7.** Rows with different superscripts (a, b, e) indicate P < 0.05. d Percentage of transferable embryos based on the

Sexed 12/24

recovered.Adapted from Soares et al. [66].Embryo production of Nelore cows (*Bos indicus*) superovulated and

Number of cows 11 11 11 ---- Total ova/embryos 10.4 ± 3.4 11.3 ± 4.4 12.4 ± 3.8 0.40 Transferable embryos (n) 8.7 ± 2.8a 4.6 ± 3.0b 6.4 ± 3.1ab 0.007

(%)d 85.9 ± 14.0a 40.7 ± 21.3c 54.2 ± 23.2b < 0.001 Freezable embryos (n) 6.9 ± 1.8a 3.2 ± 1.8b 5.4 ± 3.4ab 0.007 Freezable embryos (%)e 69.9 ± 16.8a 29.9 ± 15.5c 45.3 ± 26.6b < 0.001 Degenerate embryos 0.7 ± 0.9 1.4 ± 1.8 1.3 ± 1.7 0.43 Unfertilized oocytes 0.9 ± 1.4a 5.2 ± 3.1b 4.6 ± 2.6b 0.0003

**Table 8.** Rows with different superscripts (a, b, c) indicate P < 0.05. d Percentage of transferable embryos based on the

recovered.Adapted from Soares et al. [66].Embryo production of Holstein cows (*Bos Taurus*) superovulated and

Percentage of freezable embryos based on the number of ova/embryos

Percentage of freezable embryos based on the number of ova/embryos

Treatments P *value*

Sexed 18/30

**Treatments P value**

Sexed 18/30

and 30 h (Table 8).

number of ova/embryos recovered. e

Transferable embryos

number of ova/embryos recovered. e

inseminated in different times with conventional or sexed semen.

inseminated in different times with conventional or sexed semen.

Conventional 12/24

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 TAI (P = 0.001).

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

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 to the site of ovulation would potentially provide better results [64, 78]. Also, it is important to mention that in most of those studies, the cornual inseminations were performed by de‐ positing half of the semen straw into the right horn and the other half into the left horn. In the Sá Filho et al. [31] study, the insemination was performed with the full dose in the ute‐ rine horn ipsilateral to the expected side of ovulation. Despite these differences, similar P/AI was found when comparing inseminations performed in the horn or in the body of the ute‐ rus, which agrees with those previous results with conventional semen.

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