**2. The importance of semen quality for AI programs**

Regarding the quality of semen used in AI programs, it has been reported that differences in fertility level could be attributed to variations in sperm qualitative characteristics [11].The success of bovine AI programs largely depends on the use of good quality semen. When only high fertility bulls are used, better conception rates are achieved, which reduces costs of reproductive programs [12].

bath of 36°C [19]. Later, several other studies were performed regarding this thawing practice, in order to evaluate the effect of simultaneous thawing of semen straws on *in vivo* fertility

The Importance of Semen Quality in AI Programs and Advances in Laboratory Analyses...

http://dx.doi.org/10.5772/52022

3

Goodell [24], in a study with only 180 reproductive outcomes, reported a decrease in concep‐ tion rates of the third and fourth insemination in the sequence, when more than two straws were thawed at once. However, Kaproth et al. [26] and Dalton et al. [27] demonstrated that experienced AI technicians can simultaneously thaw multiple semen straws and inseminate up to four cows within a 20 min interval, without adverse effects on field fertility. Sprenger et al. [25] observed that an interaction of herd by sequential insemination tended to influence field fertility outcomes. In one herd, conception rates of straws number 6 and ≥ 7 were lower than conception rates of straws 1 to 5. However, in the further eleven herds evaluated, sequential insemination had no effect on conception rate. The authors concluded that, given that recommended semen handling procedures are followed, more than two straws can be

DeJarnette et al. [29,30] reviewed several studies regarding the effects of sequence of insemi‐ nation after simultaneous thawing on conception rates with data collected from about 19,000 inseminations. The combined data from several studies suggested that several straws can be thawed at once with no significant fertility concern, provided that inseminators strictly adhere to recommended semen handling procedures. The authors recommended thawing (at 35°C for a minimum of 45 seconds) only the number of straws that can be deposited within 10 to 15 minutes in the female reproductive tract (always maintaining the thermal homeostasis during this interval) to avoid semen fertility impairment. In addition, it was stated that the more important issues regarding semen handling is time, temperature, hygiene and inseminator proficiency. Technicians that fail to abide by the standard recommendations will likely realize

less than optimal conception rates irrespective of the number of straws thawed [29].

Hence, in general, the standard recommendations for cryopreserved bovine semen are (unless otherwise specified by the manufacturer): 1) to thaw no more straws than can be deposited in the female within 15 minutes between thawing and insemination, in a water-bath at 35°C for a minimum of 45 seconds, always maintaining thermal homeostasis during this interval; 2) Prevent direct straw to straw contact during the thaw process; 3) Implement appropriate thermal and hygienic protection procedures to maintain thermal homeostasis and cleanliness

Still, in a recent study, Oliveira et al. [28] observed that pregnancy rate was affected by sequence of insemination, depending on which bull was utilized in a timed AI program. In this experi‐ ment, groups of ten semen straws (0.5 mL) were simultaneously thawed at 36°C. After 30 seconds, semen straws were removed (one straw at a time) from water-bath and subsequently deposited in the cows for AI. One semen straw was used for each cow, in the same sequence that they were removed from water-bath. All animals utilized in the study were Nelore cows (n = 944). The inseminations were performed with semen from three Angus bulls, during Brazilian summer season (breeding season for beef cattle). Timed AI procedures were per‐ formed in a covered and protected area. The results demonstrated that one of the three sires had reduced fertility for inseminations performed with the group of straws associated with

following AI [23-28].

thawed at once without compromising semen fertility.

during gun assembly and transport to the cow [29].

Individual bulls may differ in their ability to fertilize oocytes and/or to develop to blastocyst stages after *in vitro* fertilization (IVF) procedures [12-18]. In addition, different sires and/or batches may differ in the individual response to induction of *in vitro* sperm capacitation methods, [14] and in the response to acrosomal mantainence after *in vitro* incubation [19].

Moreover, the bull influence is an important factor affecting *in vivo* reproductive outcomes [8,11, 20,21]. Ward et al. [20] demonstrated that kinetics of embryo development post insemi‐ nation may vary between bulls. Andersson et al. [21] observed a high variability in fertility among bulls using different sperm concentrations per dose at AI. Sá Filho et al. [8] reported a high variation in conception rates depending on the bull utilized in a Timed-AI program. Moreover, Oliveira et al. [10] observed that the sire with numerically lower field fertility also presented inferior semen quality based on the several *in vitro* sperm characteristics assessed.

Furthermore, semen handling (and/or semen thawing protocol) might also be an important factor influencing in semen quality and, therefore, in AI results. Hence, it is deemed necessary to alert to the practice of simultaneous thawing of multiple semen straws at the moment of AI.

For instance, the Brazilian Association of Artificial Insemination recommends, for bovine AI, the thawing procedure of a single frozen semen straw (0.5 mL) in water bath unit at a tem‐ perature of 35 to 37°C for 30 seconds [22]. However, the large size of breeding herds using Timed-AI protocols in Brazil have resulted in the routine practice of thawing multiple straws simultaneously in the same water-bath unit to increase the convenience of semen handling and the number of inseminations in a short period.

Because the size of breeding herds continues to increase and the use of estrus synchronization (as well as the fixed-time artificial insemination protocols) becomes more frequent worldwide, there are increasing probabilities that several cows will be inseminated on the same day. Hence, several inseminators have used the practice of thawing, simultaneously, more than one straw of semen in the same thawing-bath unit to increase the convenience of semen handling. However, under these conditions, some straws remain in the thawing bath while insemination occurs. Consequently, the thermal environment of the water bath could have some influence in sperm viability and fertility.

With this concern Brown et al. [19] demonstrated, in a laboratory study, that semen straws must be agitated immediately after plunging to prevent direct contact among semen doses and refreezing during the thaw process. In this case, the simultaneous thawing of multiple straws had no effect on percentage of motile spermatozoa and acrosomal integrity when up to ten 0.5-mL semen straws were simultaneously thawed in a thermostatically controlled thawing bath of 36°C [19]. Later, several other studies were performed regarding this thawing practice, in order to evaluate the effect of simultaneous thawing of semen straws on *in vivo* fertility following AI [23-28].

**2. The importance of semen quality for AI programs**

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

reproductive programs [12].

Regarding the quality of semen used in AI programs, it has been reported that differences in fertility level could be attributed to variations in sperm qualitative characteristics [11].The success of bovine AI programs largely depends on the use of good quality semen. When only high fertility bulls are used, better conception rates are achieved, which reduces costs of

Individual bulls may differ in their ability to fertilize oocytes and/or to develop to blastocyst stages after *in vitro* fertilization (IVF) procedures [12-18]. In addition, different sires and/or batches may differ in the individual response to induction of *in vitro* sperm capacitation methods, [14] and in the response to acrosomal mantainence after *in vitro* incubation [19].

Moreover, the bull influence is an important factor affecting *in vivo* reproductive outcomes [8,11, 20,21]. Ward et al. [20] demonstrated that kinetics of embryo development post insemi‐ nation may vary between bulls. Andersson et al. [21] observed a high variability in fertility among bulls using different sperm concentrations per dose at AI. Sá Filho et al. [8] reported a high variation in conception rates depending on the bull utilized in a Timed-AI program. Moreover, Oliveira et al. [10] observed that the sire with numerically lower field fertility also presented inferior semen quality based on the several *in vitro* sperm characteristics assessed.

Furthermore, semen handling (and/or semen thawing protocol) might also be an important factor influencing in semen quality and, therefore, in AI results. Hence, it is deemed necessary to alert to the practice of simultaneous thawing of multiple semen straws at the moment of AI.

For instance, the Brazilian Association of Artificial Insemination recommends, for bovine AI, the thawing procedure of a single frozen semen straw (0.5 mL) in water bath unit at a tem‐ perature of 35 to 37°C for 30 seconds [22]. However, the large size of breeding herds using Timed-AI protocols in Brazil have resulted in the routine practice of thawing multiple straws simultaneously in the same water-bath unit to increase the convenience of semen handling

Because the size of breeding herds continues to increase and the use of estrus synchronization (as well as the fixed-time artificial insemination protocols) becomes more frequent worldwide, there are increasing probabilities that several cows will be inseminated on the same day. Hence, several inseminators have used the practice of thawing, simultaneously, more than one straw of semen in the same thawing-bath unit to increase the convenience of semen handling. However, under these conditions, some straws remain in the thawing bath while insemination occurs. Consequently, the thermal environment of the water bath could have some influence

With this concern Brown et al. [19] demonstrated, in a laboratory study, that semen straws must be agitated immediately after plunging to prevent direct contact among semen doses and refreezing during the thaw process. In this case, the simultaneous thawing of multiple straws had no effect on percentage of motile spermatozoa and acrosomal integrity when up to ten 0.5-mL semen straws were simultaneously thawed in a thermostatically controlled thawing

and the number of inseminations in a short period.

in sperm viability and fertility.

Goodell [24], in a study with only 180 reproductive outcomes, reported a decrease in concep‐ tion rates of the third and fourth insemination in the sequence, when more than two straws were thawed at once. However, Kaproth et al. [26] and Dalton et al. [27] demonstrated that experienced AI technicians can simultaneously thaw multiple semen straws and inseminate up to four cows within a 20 min interval, without adverse effects on field fertility. Sprenger et al. [25] observed that an interaction of herd by sequential insemination tended to influence field fertility outcomes. In one herd, conception rates of straws number 6 and ≥ 7 were lower than conception rates of straws 1 to 5. However, in the further eleven herds evaluated, sequential insemination had no effect on conception rate. The authors concluded that, given that recommended semen handling procedures are followed, more than two straws can be thawed at once without compromising semen fertility.

DeJarnette et al. [29,30] reviewed several studies regarding the effects of sequence of insemi‐ nation after simultaneous thawing on conception rates with data collected from about 19,000 inseminations. The combined data from several studies suggested that several straws can be thawed at once with no significant fertility concern, provided that inseminators strictly adhere to recommended semen handling procedures. The authors recommended thawing (at 35°C for a minimum of 45 seconds) only the number of straws that can be deposited within 10 to 15 minutes in the female reproductive tract (always maintaining the thermal homeostasis during this interval) to avoid semen fertility impairment. In addition, it was stated that the more important issues regarding semen handling is time, temperature, hygiene and inseminator proficiency. Technicians that fail to abide by the standard recommendations will likely realize less than optimal conception rates irrespective of the number of straws thawed [29].

Hence, in general, the standard recommendations for cryopreserved bovine semen are (unless otherwise specified by the manufacturer): 1) to thaw no more straws than can be deposited in the female within 15 minutes between thawing and insemination, in a water-bath at 35°C for a minimum of 45 seconds, always maintaining thermal homeostasis during this interval; 2) Prevent direct straw to straw contact during the thaw process; 3) Implement appropriate thermal and hygienic protection procedures to maintain thermal homeostasis and cleanliness during gun assembly and transport to the cow [29].

Still, in a recent study, Oliveira et al. [28] observed that pregnancy rate was affected by sequence of insemination, depending on which bull was utilized in a timed AI program. In this experi‐ ment, groups of ten semen straws (0.5 mL) were simultaneously thawed at 36°C. After 30 seconds, semen straws were removed (one straw at a time) from water-bath and subsequently deposited in the cows for AI. One semen straw was used for each cow, in the same sequence that they were removed from water-bath. All animals utilized in the study were Nelore cows (n = 944). The inseminations were performed with semen from three Angus bulls, during Brazilian summer season (breeding season for beef cattle). Timed AI procedures were per‐ formed in a covered and protected area. The results demonstrated that one of the three sires had reduced fertility for inseminations performed with the group of straws associated with the longest interval from thawing to AI. However, semen from the other two bulls was not significantly different with respect to field fertility for any straw group (Straw Group 1: inseminations with 1st, 2nd and 3rd straws of the sequence; Straw Group 2: inseminations with 4th, 5th and 6th straws of the sequence; Straw Group 3: inseminations with 7th, 8th, 9th and 10th straws of the sequence). The mean time (±SD) of straws remaining in the thawing bath were 01:30 ± 00:51 for Straw Group 1, 03:36 ± 01:10 for Straw Group 2 and 06:13 ± 01:44 min for Straw Group 3. There was an interaction between sire and Straw Group (Conception rate of Sire 1: Straw Group 1 = 58.1%, Straw Group 2 = 60.2% and Straw Group 3 = 35.3%, P < 0.05; Conception rate of Sire 2: Straw Group 1 = 40.2%, Straw Group 2 = 50.5% and Straw Group 3 = 51.7%, P > 0.05; Conception rate of Sire 3: Straw Group 1 = 59.8%, Straw Group 2 = 51.0% and Straw Group 3 = 48.6%, P > 0.05). Overall conception rate of cows inseminated with first straw in the sequence (Straw 1) was 58% and of cows inseminated with tenth straw in the sequence (Straw 10) was 44% (P > 0.05). According to the results, semen fertility of some sires appeared to be more negatively affected by sequence of insemination than others. However, because the high environmental temperature during the field experiment may have potentiated the effects of incubation time on semen quality, the possibility that the thermal environment of thawing bath could have interfered on sperm fertility (mainly of bull that presented reduced conception rate associated to sequence of insemination), was considered. In summary, it was stated that the number of straws that can be simultaneously thawed without compromising semen fertility seems to vary for each bull. Unfortunately, the laboratory analyses did not clarified the effect of interaction between sire and straw group observed in field experiment of this respective study [28]. Thus, the reason why semen from some bulls seems to be more suscep‐ tible to specific thawing environments and/or procedures remained to be elucidated. The authors concluded that sequence of insemination after simultaneous thawing of multiple semen straws might affect fertility outcomes, depending on the sire utilized in the reproductive program. Hence, under similar environmental conditions, 10 semen straws should not be simultaneously thawed, because it could affect conception rates, according to the semen that is being used. Therefore, in similar routine procedures of timed AI programs consisting of large herds, it seems more cautious to not exceed the number of six semen straws for simul‐ taneous thawing [28].

post-thaw fertility maintenance. In view of the fact that the studies of Lee et al. [23] and Oliveira et al. [28] were performed during warm seasons of tropical (or subtropical) environments, it can be suggested that greater sequences of insemination might compromise conception rates when associated with the effects of higher ambient temperatures and/or solar exposure.

The Importance of Semen Quality in AI Programs and Advances in Laboratory Analyses...

http://dx.doi.org/10.5772/52022

5

Given the above observations, even though that many factors related to semen quality might influence AI outcomes, it is noteworthy that the use of high fertility bulls reduces the chances of field fertility impairment. Hence, an adequate evaluation of semen quality may reduce the effect of sire on reproductive outcomes, which is commonly observed in field trials. Thus, since a proper prediction of bull fertility is increasingly required, we consider appropriate to review the correlations between field fertility and *in vitro* sperm characteristics assessed by classical

**3. Correlation between** *in vitro* **sperm characteristics and** *in vivo* **bull**

Nowadays, many classical and modern methods have been used for laboratory assessment of *in vitro* semen characteristics following cryopreservation with the main purpose of predicting

Among the several sperm characteristics evaluated by laboratory techniques, sperm motility [33,40,41], morphology [42,43] and plasma membrane integrity [11,35,36,38] are the most used laboratory tests for assessing *in vitro* semen quality. However, the results of such assays do not

In this sense, the relationship of *in vitro* semen characteristics and *in vivo* sire fertility has been the subject of much study [12,41,44,45-47]. Nevertheless, substantial variations are commonly observed in different experiments and low correlations are usually detected when single *in vitro* sperm characteristics are isolated compared to the field fertility [12,44]. Until now, the most efficient and accurate method to estimate the fertility of a particular bull is to accomplish the field fertility tests [44], which is very laborious, expensive and time consuming [46].

Alternatively, embryo culture techniques allow exploring *in vitro* bull fertility. The employ‐ ment of such techniques has provided interesting but contradictory results regarding corre‐ lations between embryo *in vitro* embryo production (IVP) and *in vivo* bull fertility. Although positive correlations between IVP results and field fertility has been reported for some authors [12,14,16, 17, 20,46,48], other studies did not confirm the positive high correlations between *in vitro* fertilization (IVF) outcomes and *in vivo* fertility of evaluated sires [49,50,51]. However, Sudano et al*.* [12] recently demonstrated that it is possible to estimate bull fertility based on

Although interesting, it is still precipitated to ensure that the individual ability of fertilizing oocytes *in vitro* is a useful parameter for predicting *in vivo* bull fertility following AI. Hence, according to Ward et al. [20], a range of protocol variations among different IVP laboratories, the low repeatability in the results, as well as the various factors that may affect IVP outcomes,

and modern semen analyses.

the fertility potential of a semen sample [11, 31-39].

always correlate with the real fertility of a semen sample [12,44].

IVF outcomes, using a Bayesian statistical inference model.

**fertility**

Similarly, Lee et al. [23] had previously reported that sequence of insemination may influence conception rates when up to four straws were thawed at once. Although all inseminations (n = 89) occurred within recommended time constraints (i.e., within the limit of 15 minutes between thawing to AI), the loaded AI guns were exposed to direct solar radiation (in a tropical environment; Hawaii) during transport from thawing-bath to cow. The data suggested that the thermal insult might had reflected in a linear reduction in conception rates from the first (48%) through the forth (25%) gun used in sequence [23].

Thus, an important consideration to be made is the possibility of a significant interaction between ambient temperature and interval to semen deposition. According to Shepard (unpublished; cited by [29]), an interaction of ambient temperature and interval to semen deposition might occur due to extended thaw duration (>10 min) when ambient temperatures are above 17°C, suggesting that higher environmental temperatures may be problematic to post-thaw fertility maintenance. In view of the fact that the studies of Lee et al. [23] and Oliveira et al. [28] were performed during warm seasons of tropical (or subtropical) environments, it can be suggested that greater sequences of insemination might compromise conception rates when associated with the effects of higher ambient temperatures and/or solar exposure.

the longest interval from thawing to AI. However, semen from the other two bulls was not significantly different with respect to field fertility for any straw group (Straw Group 1: inseminations with 1st, 2nd and 3rd straws of the sequence; Straw Group 2: inseminations with 4th, 5th and 6th straws of the sequence; Straw Group 3: inseminations with 7th, 8th, 9th and 10th straws of the sequence). The mean time (±SD) of straws remaining in the thawing bath were 01:30 ± 00:51 for Straw Group 1, 03:36 ± 01:10 for Straw Group 2 and 06:13 ± 01:44 min for Straw Group 3. There was an interaction between sire and Straw Group (Conception rate of Sire 1: Straw Group 1 = 58.1%, Straw Group 2 = 60.2% and Straw Group 3 = 35.3%, P < 0.05; Conception rate of Sire 2: Straw Group 1 = 40.2%, Straw Group 2 = 50.5% and Straw Group 3 = 51.7%, P > 0.05; Conception rate of Sire 3: Straw Group 1 = 59.8%, Straw Group 2 = 51.0% and Straw Group 3 = 48.6%, P > 0.05). Overall conception rate of cows inseminated with first straw in the sequence (Straw 1) was 58% and of cows inseminated with tenth straw in the sequence (Straw 10) was 44% (P > 0.05). According to the results, semen fertility of some sires appeared to be more negatively affected by sequence of insemination than others. However, because the high environmental temperature during the field experiment may have potentiated the effects of incubation time on semen quality, the possibility that the thermal environment of thawing bath could have interfered on sperm fertility (mainly of bull that presented reduced conception rate associated to sequence of insemination), was considered. In summary, it was stated that the number of straws that can be simultaneously thawed without compromising semen fertility seems to vary for each bull. Unfortunately, the laboratory analyses did not clarified the effect of interaction between sire and straw group observed in field experiment of this respective study [28]. Thus, the reason why semen from some bulls seems to be more suscep‐ tible to specific thawing environments and/or procedures remained to be elucidated. The authors concluded that sequence of insemination after simultaneous thawing of multiple semen straws might affect fertility outcomes, depending on the sire utilized in the reproductive program. Hence, under similar environmental conditions, 10 semen straws should not be simultaneously thawed, because it could affect conception rates, according to the semen that is being used. Therefore, in similar routine procedures of timed AI programs consisting of large herds, it seems more cautious to not exceed the number of six semen straws for simul‐

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

Similarly, Lee et al. [23] had previously reported that sequence of insemination may influence conception rates when up to four straws were thawed at once. Although all inseminations (n = 89) occurred within recommended time constraints (i.e., within the limit of 15 minutes between thawing to AI), the loaded AI guns were exposed to direct solar radiation (in a tropical environment; Hawaii) during transport from thawing-bath to cow. The data suggested that the thermal insult might had reflected in a linear reduction in conception rates from the first

Thus, an important consideration to be made is the possibility of a significant interaction between ambient temperature and interval to semen deposition. According to Shepard (unpublished; cited by [29]), an interaction of ambient temperature and interval to semen deposition might occur due to extended thaw duration (>10 min) when ambient temperatures are above 17°C, suggesting that higher environmental temperatures may be problematic to

(48%) through the forth (25%) gun used in sequence [23].

taneous thawing [28].

Given the above observations, even though that many factors related to semen quality might influence AI outcomes, it is noteworthy that the use of high fertility bulls reduces the chances of field fertility impairment. Hence, an adequate evaluation of semen quality may reduce the effect of sire on reproductive outcomes, which is commonly observed in field trials. Thus, since a proper prediction of bull fertility is increasingly required, we consider appropriate to review the correlations between field fertility and *in vitro* sperm characteristics assessed by classical and modern semen analyses.
