- Email: [email protected]
Comparison of pregnancy outcomes using either an Ovsynch or a Cosynch protocol for the first timed AI with liquid or frozen semen in lactating dairy cows
Theriogenology 107 (2018) 21e26
Contents lists available at ScienceDirect
Theriogenology journal homepage: www.theriojournal.com
Comparison of pregnancy outcomes using either an Ovsynch or a Cosynch protocol for the first timed AI with liquid or frozen semen in lactating dairy cows S. Borchardt a, L. Schüller a, L. Wolf a, C. Wesenauer b, W. Heuwieser a, * a b
€t Berlin, Koenigsweg 65, 14163 Berlin, Germany Clinic for Animal Reproduction, Faculty of Veterinary Medicine, Freie Universita RinderAllianz, Am Bullenberg 1, 17348 Woldegk, Germany
a r t i c l e i n f o
a b s t r a c t
Article history: Received 10 June 2017 Received in revised form 16 October 2017 Accepted 16 October 2017
The objective of this study was to evaluate fertility to the first timed AI (TAI) using either liquid semen or frozen semen after an Ovsynch or a Cosynch protocol in lactating dairy cows. The hypothesis was that there is an increase in fertility to the first TAI when cows are inseminated with liquid semen compared to that when frozen semen is used in a Cosynch protocol. Lactating dairy cows (n ¼ 1724; 540 primiparous, 1184 multiparous) from 9 commercial dairy farms were enrolled on a weekly basis to facilitate first timed AI. Two experiments were conducted. In experiment 1, all cows received GnRH, 7 d later PGF2a, and then received one of the following treatments: 1) GnRH þ TAI with liquid semen 56 h after PGF2a; 2) GnRH þ TAI with frozen semen 56 h after PGF2a; 3) GnRH 56 h after PGF2a þ TAI with liquid semen 12 e16 h after the second GnRH; 4) GnRH 56 h after PGF2a þ TAI with frozen semen 12e16 h after the second GnRH. In experiment 2, all cows received GnRH, 7 d later PGF2a, and then received treatments 3 or 4 as described for experiment 1. Number of sperm per straw was 20 106 sperm/straw and 10 106 sperm/straw for frozen and liquid semen, respectively. Pregnancy diagnosis was performed by ultrasound scanning at 39 d after TAI. In experiment 1 (n ¼ 1263), there was an interaction of semen preservation method by TAI protocol. Cows inseminated with liquid semen concurrently with the second GnRH (Cosynch-56) achieved greater pregnancy per AI (P/AI) than cows inseminated with frozen semen using the same synchronization protocol (20.0% vs. 27.5%; P ¼ 0.032). There was no effect of semen preservation method (liquid semen 32.3% vs. frozen semen 28.6%; P ¼ 0.330) when cows were inseminated approximately 16 h after the second GnRH injection (Ovsynch-56). Parity affected P/AI with primiparous having a greater P/AI than multiparous cows (34.8% vs. 20.2%; P ¼ 0.001). In experiment 2 (n ¼ 377), there was no effect of semen preservation method (liquid semen 26.5% vs. frozen semen 25.5%; P ¼ 0.846) when cows were inseminated approximately 16 h after the second GnRH injection (Ovsynch56). Parity affected P/AI with primiparous having a greater P/AI than multiparous cows (37.0% vs. 17.3%; P ¼ 0.001). The results of this study provide evidence that liquid semen achieved greater P/AI in a TAI protocol with a long time interval between insemination and ovulation (Cosynch-56) compared with frozen semen indicating that liquid semen might have a longer viability in the reproductive tract. © 2017 Elsevier Inc. All rights reserved.
Keywords: Liquid semen Frozen semen Ovsynch Cosynch
1. Introduction Long-term storage of semen in liquid nitrogen using plastic straws has been one of the key factors for the widespread adoption of artificial insemination (AI) in the dairy industry [1]. In 2007, about 72% of the dairy operations in the U.S. were using frozen
* Corresponding author. E-mail address: [email protected] (W. Heuwieser). https://doi.org/10.1016/j.theriogenology.2017.10.026 0093-691X/© 2017 Elsevier Inc. All rights reserved.
semen for AI [2]. The process of freezing and thawing, however, irreversibly affects a significant proportion of sperm with typical estimates of 50% survival postthawing [3,4]. This issue might be resolved by using liquid semen for AI. Liquid semen has compelling advantages such as reduced insemination doses [5,6], less damage to sperm [7], more widespread use of genetically superior sires, limiting the negative effect of sexing technology [6], lower cost of storage, and simple handling [8]. In contrast, the major disadvantage for liquid semen is its limited fertile life span of approximately 3 days [8]. Liquid semen only accounts for 5% of worldwide AI [9],
22
S. Borchardt et al. / Theriogenology 107 (2018) 21e26
predominantly in seasonal calving dairy systems such as New Zealand, Australia, and Ireland. The use and production of liquid semen can only be justified by the increase in fertility when compared with frozen semen. Synchronization protocols for timed artificial insemination (TAI) are widely adopted in the dairy industry [10]. The most common synchronization protocol is a 7 d Ovsynch [11]. The interval from induction of ovulation to insemination influences fertility of dairy cows. Pursley et al. [12] demonstrated maximum pregnancy and calving rates when cows received TAI approximately 16 h after the second GnRH injection (GnRH2). Prolonged time from insemination to ovulation (>24 h) appears to reduce pregnancy per AI (P/AI) for cows inseminated at the onset of estrus [13,14] or at the time of the GnRH2 mediated LH surge [12,15]. Decreased fertilization rates have been reported when cows were inseminated at the onset of estrus compared with breeding 12 or 24 h later [14]. A loss of sperm viability is likely responsible for the declines in fertilization rate and P/AI that have been observed in several [12,14,16] but not all [17] studies that examined extended intervals between AI and ovulation. Despite these results, some dairy farms utilize protocols in which TAI is done concurrently with GnRH2 of the 7 d Ovsynch protocol, i.e., Cosynch protocol [18]. This protocol requires one less animal intervention thus decreasing the number of potentially stressful events and lock-up times as well as labor costs. For liquid semen a higher viability in the female reproductive tract compared to cryopreserved semen has been postulated [19]. Bucher et al. [20] obtained similar P/AI in beef cows using liquid semen (n ¼ 736; 51.5%) at a concentration of 3 106 sperm/straw compared with frozen semen (n ¼ 719; 50.4%) at a concentration of 20 106 sperm/straw. In another study using beef cows, liquid semen achieved greater P/AI (n ¼ 430; 59.9%) compared with frozen semen (n ¼ 408; 49.4%) using the same concentration of 25 106 sperm/ straw in a TAI protocol [7]. In lactating dairy cows using liquid semen might be more convenient for TAI regarding the duration of the AI process because multiple inseminations can be performed more efficiently as there is no need for thawing. Controlled randomized trials (RCT) comparing liquid and frozen semen in TAI protocols for lactating dairy cows, however, are missing. Therefore, the objective of this study was to compare liquid and frozen semen using either an Ovsynch or a Cosynch protocol for the first TAI in a 2 2 factorial design. The main hypothesis of the present study was that there is an increase in P/AI for the first TAI when cows are inseminated with liquid semen compared with frozen semen in a Cosynch protocol. Additionally, we hypothesized that using an Ovsynch protocol, liquid semen achieves comparable P/AI compared with frozen semen. 2. Material and methods The experimental procedures reported herein were conducted with the approval of the Institutional Animal Care and Use Committee of the Freie Universit€ at Berlin. 2.1. Farms and animals This experiment was performed on 9 commercial dairy farms in Mecklenburg-Vorpommern, Germany from April 2016 to October 2016. Lactating dairy cows (n ¼ 1724; 540 primiparous, 1184 multiparous) were housed in free-stall facilities and had ad libitum access to fresh feed and water. Diets were typical for MecklenburgVorpommern, using corn silage as the major forage and balanced by a professional nutritional consultant for protein, vitamins, and minerals. Cows were fed a total mixed ration twice daily that consisted of corn silage and grass silage as forage with a corn, soybean meal, and canola meal-based concentrate. All total mixed
rations were balanced to meet or exceed minimum nutritional requirements for dairy cows [21]. Cows were milked either 2 (3 farms) or 3 (6 farms) times daily. Lists for scheduled injections (completed by AI personnel) and pregnancy examination (completed by the herd veterinarian) for individual cows were generated weekly using a commercial on-farm software program (5 farms used HerdeW, version 5.9, dsp-Agrosoft Ltd., Ketzin, Germany; 1 farm used DairyPlan C21, GEA, Düsseldorf, Germany; 1 farm used Dairy Comp 305; Valley Agricultural Software, Tulare, USA; 2 farms used FULLEXPERT, Lemmer-Fullwood Ltd., Lohmar, Germany). These programs were also used to track and record reproductive outcomes and individual cow events for each cow enrolled in the experiment. 2.2. Experimental design This study was designed as a controlled randomized study using a 2 2 factorial design (experiment 1). The first main effect was the type of semen preservation method used for TAI (i.e., conventional frozen semen or liquid semen). The type of semen used for TAI changed every other week on each farm (i.e., week 1: frozen semen; week 2: liquid semen; week 3: frozen semen). The second main effect was the time of insemination relative to the second GnRH injection in a 7 d TAI protocol. Cows were inseminated either 12e16 h after the second GnRH injection (Ovsynch-56) or concurrently with the second GnRH injection (Cosynch-56). Cohorts of cows were randomly assigned to 1 of 2 synchronization protocol on a weekly basis to facilitate first postpartum TAI (i.e., Ovsynch-56 or Cosynch-56). Animals were assigned to synchronization protocols based on the last digit of their unique 10-digit animal identification number. Odd-numbered cows were assigned to Ovsynch-56 and even-numbered cows were assigned to Cosynch-56. In September 2016, we compared P/AI between the Cosynch-56 and the Ovsynch-56 using pregnancy information of approximately 800 TAI. There was a marked reduction in P/AI for cows inseminated with frozen semen concurrently with GnRH2. In order to prevent pregnancy losses due to the synchronization protocol enrollment of cows into the Cosynch-56 was terminated and the farms continued with Ovsynch-56 only (experiment 2). The procedures for using the 2 semen preservation methods continued as described with type of semen used for TAI changing every other week. 2.3. Reproductive management of the farms None of the farms used a presynchronization protocol before the first TAI. Reproductive management of the first postpartum AI (i.e., VWP, DIM at enrollment) is summarized in Table 1. Days in milk when cows received the first GnRH injection represented study day 0. As illustrated in Fig. 1, animals assigned to Ovsynch-56 (n ¼ 1054) received 2 injections of GnRH (100 mg of Gonadorelin, Gonavet Veyx, Veyx Pharma Ltd., Germany) on day 0 and 9 (56 h after PGF2a). Prostaglandin F2a (500 mg Cloprostenol, PGF Veyx forte, Veyx Pharma Ltd., Germany) was administered on day 7. TAI was performed 12e16 h after the second GnRH injection on a Friday morning. Animals assigned to Cosynch-56 (n ¼ 670) received the same injection schedule as described for Ovsynch but were inseminated concurrently with the second GnRH injection Thursday evening (Fig. 1). On each farm, a single professional AI technician performed all inseminations. Pregnancy diagnosis was performed by transrectal ultrasonography. Pregnancy status was evaluated by the herd veterinarian 39 d after TAI. A positive pregnancy diagnosis was based on visualization of an embryo with a heartbeat.
S. Borchardt et al. / Theriogenology 107 (2018) 21e26
23
Table 1 Herd characteristics from the 9 participating dairy farms. Parameter
Herd size, n Enrolled cows, n 305d Milk yield, kg VWPa, d 21d PRb, % DIM at enrollmentc, d a b c
Farm number 1
2
3
4
5
6
7
8
9
929 143 9200 40 14 64
620 195 10,300 40 16 61
864 205 10,200 40 15 69
1020 197 10,400 60 16 70
497 140 9950 40 16 69
1380 123 10,200 50 15 79
1400 223 10,400 40 12 62
740 179 9800 50 15 69
1080 235 9600 50 15 60
Voluntary waiting period. 21d pregnancy rate. DIM corresponding to the first day of the TAI protocol.
2.4. Bulls and semen processing procedures Sires (n ¼ 10) producing the semen for this study were predominantly genomically selected Holstein-Friesian bulls. Sires were not randomly allocated to the farms due to individual breeding objectives on the farms. Ejaculates for producing liquid and frozen semen were collected using an artificial vagina per standard operating procedure twice a week (Monday and Wednesday) between 0600 and 0800 h. For the study only ejaculates from Wednesday were considered for the use of liquid semen. Ejaculates that met the required quality standards (i.e., >70% progressive motility; > 1.5 ml volume; > 500 106 sperm) were used for semen production. All ejaculates failing to meet these criteria were excluded from the study. The amount of liquid semen produced from each ejaculate was based on the requested amount of straws for that day. The remaining semen was used for the production of frozen semen. Frozen semen was diluted using OptiXcel liquid semen extender (IMV Technologies, Normandy, France) to 20 106 sperm/straw. Liquid semen was diluted using Caprogen liquid semen extender [8] to 10 106 sperm/straw. The 0.25 mL straws were filled with semen, sealed, and dispatched to technicians in the evening of the day of collection. During transport the straws were placed in a waterproof, insulated container, and submerged in water with a temperature of 10e15 C at the beginning. Frozen semen was transported to the farms and stored using containers with liquid nitrogen. Cows inseminated using the Ovsynch-56 protocol were
inseminated with liquid semen approximately 48 h after semen collection. Cows inseminated using the Cosynch-56 protocol were inseminated using liquid semen approximately 32 h after semen collection. 2.5. Statistical analyses Breeding information (i.e., cow ID, calving date, lactation number, breeding date, sire ID) and results of pregnancy diagnosis were obtained from the on farm computer system, exported to Excel (Office 2010, Microsoft Deutschland Ltd., Munich, Germany) spreadsheets and analyzed using SPSS for Windows (version 22.0, SPSS Inc., IBM, Ehningen, Germany). The analysis of P/AI at first TAI was performed by logistic regression using the GENLINMIXED procedure of SPSS. Cow was the experimental unit. Herd was considered as a random effect. According to the model-building strategies described by Dohoo et al. [22] each parameter considered for the mixed model should be separately analyzed in an univariate model, including the parameter as a fixed factor (i.e., categorical parameter) or covariate (i.e., continuous parameter). Only parameters resulting in univariate models with P 0.2 should be included in the final mixed model. Selection of the model that best fit the data was performed by testing each effect separately in an univariate model and finding the model with the lowest value for the Akaike information criterion (AIC) using a backward elimination procedure that removed all variables with P > 0.10 from the model. We used 2 different models in order to account for experiment 1 (Cosynch-56 and Ovsynch-56) and 2 (Ovsynch-56 only). 2.5.1. Experiment 1 The initial model contained the following explanatory variables as fixed effects: semen preservation method (frozen vs. liquid semen), TAI protocol (Ovsnych-56 vs. Cosynch-56), parity (primiparous vs. multiparous), sire (sire 1 to 10), DIM at first TAI (continuous). Regardless of the significance level type of semen and TAI protocol was forced to remain in the model.
Fig. 1. Experimental design. Lactating dairy cows were assigned randomly to 1 of 2 protocols for timed artificial insemination (TAI) according to the last digit of the ear tag number (odd or even). All enrolled cows in study experiment 1 received 100 mg of GnRH at GnRH1, or GnRH2 and 500 mg of PGF2a 7 d after GnRH1. Insemination was either performed concurrently with GnRH2 (Cosynch-56) or 12e16 h after GnRH2 (Ovsynch-56). In experiment 2, insemination was performed only 12e16 h after GnRH2 (Ovsynch-56). Semen preservation method (liquid vs. frozen) used for TAI changed every other week on each farm in experiment 1 and 2.
2.5.2. Experiment 2 In experiment 2, cows were only inseminated using the Ovsynch-56. Therefore, TAI protocol was not included in the model. The initial model contained the following explanatory variables as fixed effects: semen preservation method (frozen vs. liquid semen), parity (primiparous vs. multiparous), sire (sire 1 to 10), DIM at first TAI (continuous). Regardless of the significance level type of semen was forced to remain in the model. The adjusted P/AI values provided by SPSS are given throughout the text. The unadjusted values and actual experimental units (number pregnant divided by number inseminated) are also provided in Table 2.
24
S. Borchardt et al. / Theriogenology 107 (2018) 21e26
Table 2 Comparison of pregnancy per AI (P/AI) between cows inseminated with frozen semen or liquid semen and synchronized with Ovsynch-56 or Cosynch-56 for first timed artificial insemination in experiment 1 and 2. Parameter
Cosynch-56
Ovsynch-56
Frozen semen
Liquid semen
Frozen semen
Liquid semen
20.0x 19.5 60/307
27.5y 25.1 85/339
28.6 25.9 74/285
32.3 30.1 100/332
e e e
e e e
25.5 21.4 38/177
26.5 25.0 50/200
19.5 60/307
25.1 85/339
24.2 112/462
28.2 150/532
a
Experiment 1 Least square estimateb P/AI (%) n/n Experiment 2c Least square estimateb P/AI (%) n/n Overall P/AI (%) n/n x,y
Means within the same row with different superscripts differ (P < 0.05). In experiment 1 cows were either inseminated with liquid semen or frozen semen using either a Cosynch-56 or an Ovsynch-56. b Results obtained from the GENLINMIXED procedure. c In experiment 2 cows were either inseminated with liquid semen or frozen semen using an Ovsynch-56. a
3. Results Overall, 1724 AI were performed during this study. Eighty-four cows, however, were eliminated from the trial before pregnancy diagnosis because they were sold or died. Therefore, 1640 inseminations were used for statistical analyses.
injection (Ovsynch-56). In addition, parity affected P/AI with primiparous having a greater P/AI than multiparous cows (34.8% vs. 20.2%; P ¼ 0.001). There was no interaction of parity and semen type (P ¼ 0.872) or parity and TAI protocol (P ¼ 0.168). Sire had no effect on P/AI (P ¼ 0.899). 3.2. Experiment 2
3.1. Experiment 1 In experiment 1, the information of 1263 inseminations was available. There was no difference in DIM at first TAI between cows inseminated with frozen or liquid semen (P ¼ 0.604). The number of primiparous cows inseminated with frozen or liquid semen did not differ (P ¼ 0.700). The effect of semen preservation was conditional on TAI protocol. The results are summarized in Table 2 and Fig. 2. Cows inseminated with liquid semen concurrently with GnRH2 (Cosynch-56) achieved greater P/AI than cows inseminated with frozen semen using the same synchronization protocol (20.0% vs. 27.5%; P ¼ 0.032). There was no effect of semen preservation method (liquid semen 32.3% vs. frozen semen 28.6%; P ¼ 0.330) when cows were inseminated approximately 16 h after GnRH2
40
Pregnancy per AI, %
35 30 25 20 15 10 5 0 Frozen semen Liquid semen Frozen semen Liquid semen (n = 307) (n = 339) (n = 285) (n = 332) Cosynch-56
Ovsynch-56
Fig. 2. Comparison of pregnancy per AI between cows inseminated with frozen semen or liquid semen and synchronized either with Ovsynch-56 or Cosynch-56 for the first timed artificial insemination in experiment 1 using least square estimates (mean ± SEM) from the GENLINMIXED procedure.
In experiment 2, the information of 377 inseminations was available. There was no difference in DIM at first TAI between cows inseminated with frozen or liquid semen (P ¼ 0.532). The number of primiparous cows inseminated was greater (P ¼ 0.010) for cows inseminated with liquid semen (71/200) compared with cows inseminated with frozen semen (41/177). There was no effect of semen preservation method on P/AI (P ¼ 0.846). Parity affected P/AI with primiparous having a greater P/AI than multiparous cows (37.0% vs. 17.3%; P ¼ 0.001). There was no interaction of parity and semen type (P ¼ 0.929). Results for experiment 2 are summarized in Table 2. 4. Discussion This is the first randomized, controlled study to directly compare liquid semen with frozen semen in lactating dairy cows using timed AI protocols. Liquid semen achieved greater P/AI in a TAI protocol with a long time interval (~24 h) between insemination and ovulation (Cosynch-56) compared with frozen semen indicating that liquid semen might have a longer viability in the reproductive tract. There was no effect of semen preservation method on P/AI in a TAI protocol with a time interval from insemination to expected ovulation of 12e16 h (Ovsynch-56). While there is some evidence in beef cows indicating superior fertility of liquid semen [7,20,23], there is a dearth of published information on the fertility of liquid semen compared to frozen semen in dairy cows. Liquid semen processing yields more doses per ejaculate, thereby, facilitating the utilization of genetically superior sires. This is particularly beneficial for young genomically selected sires as these sires are in high demand but produce lower semen volumes in comparison to mature bulls [24]. In the absence of an improvement in P/AI of liquid semen over conventional frozen semen and with sufficient semen availability, there is little incentive for the dairy managers to use this product. For the AI companies a product with a limited shelf-life requiring a more sophisticated logistic is of limited value unless there is a higher
S. Borchardt et al. / Theriogenology 107 (2018) 21e26
conception risk. A recent study from Ireland evaluated the effect of 3 different semen diluents on P/AI using liquid semen in grazing dairy cows [25]. Overall, there was a decline in fertility for liquid semen, as measured by calving rate, with prolonged interval from semen collection until insemination. A reduced dose of liquid semen (5 106 sperm/straw) diluted in Caprogen or INRA96 resulted, however, in a similar fertility compared with frozen semen (15 106 sperm/straw). Our results indicated a superior fertility of liquid semen compared with frozen semen using only timed AI in high producing dairy cows in a confinement system. Results from these 2 studies can hardly be compared as they differ in sperm dose per straw for liquid and frozen semen, semen diluents, housing of cows, and method of insemination (i.e., AI after estrous detection vs. timed AI). Using liquid semen in timed AI protocols in conjunction with reduced sperm concentration and rapid overnight semen delivery may provide an opportunity to enhance utilization efficiency of dairy sires that are in high demand and short supply. Liquid semen minimizes time and labor necessary on the day of breeding because thawing of semen is not required. The freezethaw process can be an opportunity for human error with a negative impact on semen quality. Compared with frozen semen, the positive effect of liquid semen on P/AI was mainly attributed to the poor performance of frozen semen in the Cosynch-56 rather than the Ovsynch-56. Although there was a numerical increase of 4% points in P/AI in favor of the liquid semen considering the Ovsynch-56 the power (41.4%) of this study was not enough to detect a significant difference. Among other factors, success of timed AI programs depends on proper timing of insemination relative to ovulation [11]. For the standard Ovsynch program, with 7 days between the initial GnRH and PGF2a injection, maximum pregnancy risk in dairy cows can be achieved by administering the final GnRH 56 h after PGF2a and performing AI 16 h later [12]. The timing of AI, relative to the expected time of ovulation, is important for P/AI performing either AI after estrous detection [13] or timed AI [15]. The reduction in P/AI following Cosynch-56 is physiologically plausible based on the limited life span of sperm and the probable reduction in fertilization rate after a prolonged time from placing frozen thawed semen in the reproductive tract until ovulation [14]. This is in agreement with our results and supported by other studies [11,12,15] using frozen semen as well. The improvement in fertility, as measured by P/AI, by using Ovsynch-56 compared with Cosynch, is likely to offset an increase in labor costs for implementation of this protocol [15]. While this is applicable to frozen semen, our results indicate that in a Cosynch-56 using liquid semen dairy managers can save labor costs without affecting P/AI. In our study, primiparous cows had greater P/AI compared with multiparous cows irrespective of TAI protocol or semen preservation method. There are conflicting reports on the effect of parity on P/AI with the majority of studies showing a greater conception risk in primiparous cows [26e29] but some others showing no effect of parity [30e32]. This difference might be due to a parity specific response during the synchronization protocol [33]. In primiparous cows, a major risk factor for poor reproductive performance in a TAI protocol is the lack of a functional corpus luteum at the first GnRH injection. Therefore, primiparous cows benefit more from presynchronization using GnRH as they have a greater risk for anovulation [34,35]. Recent research also indicates that lacking luteolytic response in multiparous cows to a single prostaglandin administration in a 7 day Ovsynch protocol has a detrimental effect on P/AI [36]. For multiparous cows presynchronized with a Double-Ovsynch protocol an additional prostaglandin injection on d 8 of the breeding Ovsynch led to exceptional P/AI [32,36]. Besides parity-specific differences
25
related to successful synchronization in TAI protocols other contributors might be responsible for reduced fertility in multiparous cows such as the greater uterine size [37], greater BCS loss in early lactation [38], or a greater disease incidence in the transition period which was associated with reduced fertility later in lactation [39]. This study was initially conducted in a 2 2 factorial design to compare liquid and frozen semen using either an Ovsynch-56 or a Cosynch-56 protocol for the first TAI. After 5 months it became obvious that the Cosynch-56 was clearly inferior while using frozen semen. Therefore, we decided to terminate the Cosynch-56 to prevent economic disadvantages from the participating farms as reproductive performance is considered to be a major driver of overall farm profitability [40]. The premature termination of one study component was a compromise between the original study design and ethical considerations. We adapted to this change the experimental design by considering the 2 time periods (time period 1 Cosynch-56 and Ovsynch-56 and 2 Ovsynch-56 only) as 2 different experiments. A source of potential bias was the weekly change of semen used. This was done to improve overall compliance to a given protocol. We assumed that most farms would not be able to handle two different TAI protocols and two different semen handling methods concurrently without an increasing failure rate. Random allocation of cows into the timed AI protocol was based on the last digit of their unique 10-digit animal identification number. Ear tags were attached at birth of the calves in a consecutive order without knowledge of this study being implemented years later. Other studies randomizing cows based on their animal identification number had not been performed on the participating farms. Veterinarians conducting pregnancy diagnoses were not blinded to the study procedures and the allocation of cows to the protocols. The outcome, however, was of binary nature (i.e., pregnancy or non-pregnancy) and the veterinarians had no interest to make false-positive or false negative diagnoses. Therefore, we are comfortably sure that the results were not biased by the investigator. 5. Conclusions The results of this study provide evidence that liquid semen achieved greater P/AI in a Cosynch-56 protocol that is expected to have a longer interval between insemination and ovulation compared with frozen semen indicating that liquid semen might have a longer viability in the reproductive tract. Further research is warranted to evaluate the performance of liquid semen in cows inseminated after estrous detection with different intervals from AI to ovulation. Conflict of interest statement Neither of the authors of this paper has a financial or personal relationship with other people or organisations that could inappropriately influence or bias the content of the paper. Acknowledgements We are thankful to Veyx Pharma GmbH for providing the PGF2a and GnRH and the scientific collaboration. The authors are grateful to the owners and employees of the collaborating dairy farms for granting us access to their facilities, cows, and herd records and their wonderful support. Lisa Wolf was sponsored in part by Tiergyn e.V. Berlin.
26
S. Borchardt et al. / Theriogenology 107 (2018) 21e26
References [1] Stevenson JS. Impact of reproductive technologies on dairy food production in the dairy industry. Adv Exp Med Biol 2014;752:115e29. [2] NAHMS (national animal health monitoring service) reproductive practices on U.S. Dairy operations. 2007. [3] Gravance CG, Vishwanath R, Pitt C, Garner DL, Casey PJ. Effects of cryopreservation on bull sperm head morphometry. J Androl 1998;19:704e9. [4] Yoshida M. Conservation of sperms: current status and new trends. Anim Reprod Sci 2000;60e61:349e55. [5] Al Naib A, Ward F, Kelly AK, Wade M, Marti JI, Lonergan P. Effect of duration of storage at ambient temperature on fertilizing ability and mucus penetration ability of fresh bovine sperm. Theriogenology 2011;76:1070e5. [6] Xu ZZ. Application of liquid semen technology improves conception rate of sex-sorted semen in lactating dairy cows. J Dairy Sci 2014;97:7298e304. [7] Borges-Silva JC, Silva MR, Marinho DB, Nogueira E, Sampaio DC, Oliveira LO, et al. Cooled semen for fixed-time artificial insemination in beef cattle. Reprod Fertil Dev 2015;28:1004e8. [8] Vishwanath R, Shannon P. Storage of bovine semen in liquid and frozen state. Anim Reprod Sci 2000;62:23e53. [9] Thibier M, Wagner HG. World statistics for artificial insemination in cattle. 14th International Congress on Animal Reproduction. 2000. p. 76. [10] Caraviello DZ, Weigel KA, Fricke PM, Wiltbank MC, Florent MJ, Cook NB, et al. Survey of management practices on reproductive performance of dairy cattle on large US commercial farms. J dairy Sci 2006;89:4723e35. [11] Stevenson JS. Synchronization and artificial insemination strategies in dairy herds. Vet Clin North Am Food Anim Pract 2016;32:349e64. [12] Pursley JR, Silcox RW, Wiltbank MC. Effect of time of artificial insemination on pregnancy rates, calving rates, pregnancy loss, and gender ratio after synchronization of ovulation in lactating dairy cows. J Dairy Sci 1998;81: 2139e44. [13] Dransfield MB, Nebel RL, Pearson RE, Warnick LD. Timing of insemination for dairy cows identified in estrus by a radiotelemetric estrus detection system. J Dairy Sci 1998;81:1874e82. [14] Dalton JC, Nadir S, Bame JH, Noftsinger M, Nebel RL, Saacke RG. Effect of time of insemination on number of accessory sperm, fertilization rate, and embryo quality in nonlactating dairy cattle. J Dairy Sci 2001;84:2413e8. [15] Brusveen DJ, Cunha AP, Silva CD, Cunha PM, Sterry RA, Silva EP, et al. Altering the time of the second gonadotropin-releasing hormone injection and artificial insemination (AI) during Ovsynch affects pregnancies per AI in lactating dairy cows. J Dairy Sci 2008;91:1044e52. [16] Trimberger GW. Conception rate in dairy cattle by artificial insemination at various intervals before and after ovulation. J Dairy Sci 1944;27:659e60. [17] Portaluppi MA, Stevenson JS. Pregnancy rates in lactating dairy cows after presynchronization of estrous cycles and variations of the Ovsynch protocol. J Dairy Sci 2005;88:914e21. [18] Geary TW, Whittier JC. Effect of a timed insemination following synchronization of ovulation using the Ovsynch or Co-Synch protocol in beef cows. Prof Anim Sci 1998;14:217e20. [19] Curry MR. Cryopreservation of semen from domestic livestock. Rev Reprod 2000;5:46e52. [20] Bucher A, Kasimanickam R, Hall JB, Dejarnette JM, Whittier WD, Kahn W, et al. Fixed-time AI pregnancy rate following insemination with frozen-thawed or fresh-extended semen in progesterone supplemented CO-Synch protocol in beef cows. Theriogenology 2009;71:1180e5. [21] Nutrient requirements of dairy cattle. Washington, DC: Nat. Acad. Sci.; 2001. [22] Dohoo IR, Martin SW, Stryhn H. Veterinary epidemiologic research (2nd). Charlottetown, PEI, Canada: University of Prince Edward Island; 2009. [23] Papa PM, Maziero RD, Guasti PN, Junqueira CR, Freitas-Dell’Aqua CP, Papa FO, et al. Effect of glycerol on the viability and fertility of cooled bovine semen.
Theriogenology 2015;83:107e13. [24] Brito LF, Silva AE, Rodrigues LH, Vieira FV, Deragon LA, Kastelic JP. Effect of age and genetic group on characteristics of the scrotum, testes and testicular vascular cones, and on sperm production and semen quality in AI bulls in Brazil. Theriogenology 2002;58:1175e86. [25] Murphy EM, Murphy C, O'Meara C, Dunne G, Eivers B, Lonergan P, et al. A comparison of semen diluents on the in vitro and in vivo fertility of liquid bull semen. J Dairy Sci 2017;100:1541e54. [26] Tenhagen BA, Surholt R, Wittke M, Vogel C, Drillich M, Heuwieser W. Use of Ovsynch in dairy herds - differences between primiparous and multiparous cows. Anim Reprod Sci 2004;81:1e11. [27] Galvao KN, Sa Filho MF, Santos JE. Reducing the interval from presynchronization to initiation of timed artificial insemination improves fertility in dairy cows. J Dairy Sci 2007;90:4212e8. [28] Carvalho PD, Fuenzalida MJ, Ricci A, Souza AH, Barletta RV, Wiltbank MC, et al. Modifications to Ovsynch improve fertility during resynchronization: Evaluation of presynchronization with gonadotropin-releasing hormone 6 d before initiation of Ovsynch and addition of a second prostaglandin F2alpha treatment. J Dairy Sci 2015;98:8741e52. [29] Fuenzalida MJ, Fricke PM, Ruegg PL. The association between occurrence and severity of subclinical and clinical mastitis on pregnancies per artificial insemination at first service of Holstein cows. J Dairy Sci 2015;98:3791e805. [30] Giordano JO, Wiltbank MC, Guenther JN, Ares MS, Lopes Jr G, Herlihy MM, et al. Effect of presynchronization with human chorionic gonadotropin or gonadotropin-releasing hormone 7 days before resynchronization of ovulation on fertility in lactating dairy cows. J Dairy Sci 2012;95:5612e25. [31] Carvalho PD, Guenther JN, Fuenzalida MJ, Amundson MC, Wiltbank MC, Fricke PM. Presynchronization using a modified Ovsynch protocol or a single gonadotropin-releasing hormone injection 7 d before an Ovsynch-56 protocol for submission of lactating dairy cows to first timed artificial insemination. J Dairy Sci 2014;97:6305e15. [32] Carvalho PD, Wiltbank MC, Fricke PM. Manipulation of progesterone to increase ovulatory response to the first GnRH treatment of an Ovsynch protocol in lactating dairy cows receiving first timed artificial insemination. J Dairy Sci 2015;98:8800e13. [33] Borchardt S, Haimerl P, Pohl A, Heuwieser W. Evaluation of prostaglandin F2alpha versus prostaglandin F2alpha plus gonadotropin-releasing hormone as Presynch methods preceding an Ovsynch in lactating dairy cows: a metaanalysis. J Dairy Sci 2017;100:4065e77. [34] Souza AH, Ayres H, Ferreira RM, Wiltbank MC. A new presynchronization system (Double-Ovsynch) increases fertility at first postpartum timed AI in lactating dairy cows. Theriogenology 2008;70:208e15. [35] Herlihy MM, Giordano JO, Souza AH, Ayres H, Ferreira RM, Keskin A, et al. Presynchronization with Double-Ovsynch improves fertility at first postpartum artificial insemination in lactating dairy cows. J Dairy Sci 2012;95: 7003e14. [36] Wiltbank MC, Baez GM, Cochrane F, Barletta RV, Trayford CR, Joseph RT. Effect of a second treatment with prostaglandin F2alpha during the Ovsynch protocol on luteolysis and pregnancy in dairy cows. J Dairy Sci 2015;98:8644e54. [37] Baez GM, Barletta RV, Guenther JN, Gaska JM, Wiltbank MC. Effect of uterine size on fertility of lactating dairy cows. Theriogenology 2016;85:1357e66. [38] Carvalho PD, Souza AH, Amundson MC, Hackbart KS, Fuenzalida MJ, Herlihy MM, et al. Relationships between fertility and postpartum changes in body condition and body weight in lactating dairy cows. J Dairy Sci 2014;97: 3666e83. [39] Santos JE, Bisinotto RS, Ribeiro ES, Lima FS, Greco LF, Staples CR, et al. Applying nutrition and physiology to improve reproduction in dairy cattle. Soc Reprod Fertil Suppl 2010;67:387e403. [40] Cabrera VE. Economics of fertility in high-yielding dairy cows on confined TMR systems. Animal 2014;8(Suppl. 1):211e21.
Contents lists available at ScienceDirect
Theriogenology journal homepage: www.theriojournal.com
Comparison of pregnancy outcomes using either an Ovsynch or a Cosynch protocol for the first timed AI with liquid or frozen semen in lactating dairy cows S. Borchardt a, L. Schüller a, L. Wolf a, C. Wesenauer b, W. Heuwieser a, * a b
€t Berlin, Koenigsweg 65, 14163 Berlin, Germany Clinic for Animal Reproduction, Faculty of Veterinary Medicine, Freie Universita RinderAllianz, Am Bullenberg 1, 17348 Woldegk, Germany
a r t i c l e i n f o
a b s t r a c t
Article history: Received 10 June 2017 Received in revised form 16 October 2017 Accepted 16 October 2017
The objective of this study was to evaluate fertility to the first timed AI (TAI) using either liquid semen or frozen semen after an Ovsynch or a Cosynch protocol in lactating dairy cows. The hypothesis was that there is an increase in fertility to the first TAI when cows are inseminated with liquid semen compared to that when frozen semen is used in a Cosynch protocol. Lactating dairy cows (n ¼ 1724; 540 primiparous, 1184 multiparous) from 9 commercial dairy farms were enrolled on a weekly basis to facilitate first timed AI. Two experiments were conducted. In experiment 1, all cows received GnRH, 7 d later PGF2a, and then received one of the following treatments: 1) GnRH þ TAI with liquid semen 56 h after PGF2a; 2) GnRH þ TAI with frozen semen 56 h after PGF2a; 3) GnRH 56 h after PGF2a þ TAI with liquid semen 12 e16 h after the second GnRH; 4) GnRH 56 h after PGF2a þ TAI with frozen semen 12e16 h after the second GnRH. In experiment 2, all cows received GnRH, 7 d later PGF2a, and then received treatments 3 or 4 as described for experiment 1. Number of sperm per straw was 20 106 sperm/straw and 10 106 sperm/straw for frozen and liquid semen, respectively. Pregnancy diagnosis was performed by ultrasound scanning at 39 d after TAI. In experiment 1 (n ¼ 1263), there was an interaction of semen preservation method by TAI protocol. Cows inseminated with liquid semen concurrently with the second GnRH (Cosynch-56) achieved greater pregnancy per AI (P/AI) than cows inseminated with frozen semen using the same synchronization protocol (20.0% vs. 27.5%; P ¼ 0.032). There was no effect of semen preservation method (liquid semen 32.3% vs. frozen semen 28.6%; P ¼ 0.330) when cows were inseminated approximately 16 h after the second GnRH injection (Ovsynch-56). Parity affected P/AI with primiparous having a greater P/AI than multiparous cows (34.8% vs. 20.2%; P ¼ 0.001). In experiment 2 (n ¼ 377), there was no effect of semen preservation method (liquid semen 26.5% vs. frozen semen 25.5%; P ¼ 0.846) when cows were inseminated approximately 16 h after the second GnRH injection (Ovsynch56). Parity affected P/AI with primiparous having a greater P/AI than multiparous cows (37.0% vs. 17.3%; P ¼ 0.001). The results of this study provide evidence that liquid semen achieved greater P/AI in a TAI protocol with a long time interval between insemination and ovulation (Cosynch-56) compared with frozen semen indicating that liquid semen might have a longer viability in the reproductive tract. © 2017 Elsevier Inc. All rights reserved.
Keywords: Liquid semen Frozen semen Ovsynch Cosynch
1. Introduction Long-term storage of semen in liquid nitrogen using plastic straws has been one of the key factors for the widespread adoption of artificial insemination (AI) in the dairy industry [1]. In 2007, about 72% of the dairy operations in the U.S. were using frozen
* Corresponding author. E-mail address: [email protected] (W. Heuwieser). https://doi.org/10.1016/j.theriogenology.2017.10.026 0093-691X/© 2017 Elsevier Inc. All rights reserved.
semen for AI [2]. The process of freezing and thawing, however, irreversibly affects a significant proportion of sperm with typical estimates of 50% survival postthawing [3,4]. This issue might be resolved by using liquid semen for AI. Liquid semen has compelling advantages such as reduced insemination doses [5,6], less damage to sperm [7], more widespread use of genetically superior sires, limiting the negative effect of sexing technology [6], lower cost of storage, and simple handling [8]. In contrast, the major disadvantage for liquid semen is its limited fertile life span of approximately 3 days [8]. Liquid semen only accounts for 5% of worldwide AI [9],
22
S. Borchardt et al. / Theriogenology 107 (2018) 21e26
predominantly in seasonal calving dairy systems such as New Zealand, Australia, and Ireland. The use and production of liquid semen can only be justified by the increase in fertility when compared with frozen semen. Synchronization protocols for timed artificial insemination (TAI) are widely adopted in the dairy industry [10]. The most common synchronization protocol is a 7 d Ovsynch [11]. The interval from induction of ovulation to insemination influences fertility of dairy cows. Pursley et al. [12] demonstrated maximum pregnancy and calving rates when cows received TAI approximately 16 h after the second GnRH injection (GnRH2). Prolonged time from insemination to ovulation (>24 h) appears to reduce pregnancy per AI (P/AI) for cows inseminated at the onset of estrus [13,14] or at the time of the GnRH2 mediated LH surge [12,15]. Decreased fertilization rates have been reported when cows were inseminated at the onset of estrus compared with breeding 12 or 24 h later [14]. A loss of sperm viability is likely responsible for the declines in fertilization rate and P/AI that have been observed in several [12,14,16] but not all [17] studies that examined extended intervals between AI and ovulation. Despite these results, some dairy farms utilize protocols in which TAI is done concurrently with GnRH2 of the 7 d Ovsynch protocol, i.e., Cosynch protocol [18]. This protocol requires one less animal intervention thus decreasing the number of potentially stressful events and lock-up times as well as labor costs. For liquid semen a higher viability in the female reproductive tract compared to cryopreserved semen has been postulated [19]. Bucher et al. [20] obtained similar P/AI in beef cows using liquid semen (n ¼ 736; 51.5%) at a concentration of 3 106 sperm/straw compared with frozen semen (n ¼ 719; 50.4%) at a concentration of 20 106 sperm/straw. In another study using beef cows, liquid semen achieved greater P/AI (n ¼ 430; 59.9%) compared with frozen semen (n ¼ 408; 49.4%) using the same concentration of 25 106 sperm/ straw in a TAI protocol [7]. In lactating dairy cows using liquid semen might be more convenient for TAI regarding the duration of the AI process because multiple inseminations can be performed more efficiently as there is no need for thawing. Controlled randomized trials (RCT) comparing liquid and frozen semen in TAI protocols for lactating dairy cows, however, are missing. Therefore, the objective of this study was to compare liquid and frozen semen using either an Ovsynch or a Cosynch protocol for the first TAI in a 2 2 factorial design. The main hypothesis of the present study was that there is an increase in P/AI for the first TAI when cows are inseminated with liquid semen compared with frozen semen in a Cosynch protocol. Additionally, we hypothesized that using an Ovsynch protocol, liquid semen achieves comparable P/AI compared with frozen semen. 2. Material and methods The experimental procedures reported herein were conducted with the approval of the Institutional Animal Care and Use Committee of the Freie Universit€ at Berlin. 2.1. Farms and animals This experiment was performed on 9 commercial dairy farms in Mecklenburg-Vorpommern, Germany from April 2016 to October 2016. Lactating dairy cows (n ¼ 1724; 540 primiparous, 1184 multiparous) were housed in free-stall facilities and had ad libitum access to fresh feed and water. Diets were typical for MecklenburgVorpommern, using corn silage as the major forage and balanced by a professional nutritional consultant for protein, vitamins, and minerals. Cows were fed a total mixed ration twice daily that consisted of corn silage and grass silage as forage with a corn, soybean meal, and canola meal-based concentrate. All total mixed
rations were balanced to meet or exceed minimum nutritional requirements for dairy cows [21]. Cows were milked either 2 (3 farms) or 3 (6 farms) times daily. Lists for scheduled injections (completed by AI personnel) and pregnancy examination (completed by the herd veterinarian) for individual cows were generated weekly using a commercial on-farm software program (5 farms used HerdeW, version 5.9, dsp-Agrosoft Ltd., Ketzin, Germany; 1 farm used DairyPlan C21, GEA, Düsseldorf, Germany; 1 farm used Dairy Comp 305; Valley Agricultural Software, Tulare, USA; 2 farms used FULLEXPERT, Lemmer-Fullwood Ltd., Lohmar, Germany). These programs were also used to track and record reproductive outcomes and individual cow events for each cow enrolled in the experiment. 2.2. Experimental design This study was designed as a controlled randomized study using a 2 2 factorial design (experiment 1). The first main effect was the type of semen preservation method used for TAI (i.e., conventional frozen semen or liquid semen). The type of semen used for TAI changed every other week on each farm (i.e., week 1: frozen semen; week 2: liquid semen; week 3: frozen semen). The second main effect was the time of insemination relative to the second GnRH injection in a 7 d TAI protocol. Cows were inseminated either 12e16 h after the second GnRH injection (Ovsynch-56) or concurrently with the second GnRH injection (Cosynch-56). Cohorts of cows were randomly assigned to 1 of 2 synchronization protocol on a weekly basis to facilitate first postpartum TAI (i.e., Ovsynch-56 or Cosynch-56). Animals were assigned to synchronization protocols based on the last digit of their unique 10-digit animal identification number. Odd-numbered cows were assigned to Ovsynch-56 and even-numbered cows were assigned to Cosynch-56. In September 2016, we compared P/AI between the Cosynch-56 and the Ovsynch-56 using pregnancy information of approximately 800 TAI. There was a marked reduction in P/AI for cows inseminated with frozen semen concurrently with GnRH2. In order to prevent pregnancy losses due to the synchronization protocol enrollment of cows into the Cosynch-56 was terminated and the farms continued with Ovsynch-56 only (experiment 2). The procedures for using the 2 semen preservation methods continued as described with type of semen used for TAI changing every other week. 2.3. Reproductive management of the farms None of the farms used a presynchronization protocol before the first TAI. Reproductive management of the first postpartum AI (i.e., VWP, DIM at enrollment) is summarized in Table 1. Days in milk when cows received the first GnRH injection represented study day 0. As illustrated in Fig. 1, animals assigned to Ovsynch-56 (n ¼ 1054) received 2 injections of GnRH (100 mg of Gonadorelin, Gonavet Veyx, Veyx Pharma Ltd., Germany) on day 0 and 9 (56 h after PGF2a). Prostaglandin F2a (500 mg Cloprostenol, PGF Veyx forte, Veyx Pharma Ltd., Germany) was administered on day 7. TAI was performed 12e16 h after the second GnRH injection on a Friday morning. Animals assigned to Cosynch-56 (n ¼ 670) received the same injection schedule as described for Ovsynch but were inseminated concurrently with the second GnRH injection Thursday evening (Fig. 1). On each farm, a single professional AI technician performed all inseminations. Pregnancy diagnosis was performed by transrectal ultrasonography. Pregnancy status was evaluated by the herd veterinarian 39 d after TAI. A positive pregnancy diagnosis was based on visualization of an embryo with a heartbeat.
S. Borchardt et al. / Theriogenology 107 (2018) 21e26
23
Table 1 Herd characteristics from the 9 participating dairy farms. Parameter
Herd size, n Enrolled cows, n 305d Milk yield, kg VWPa, d 21d PRb, % DIM at enrollmentc, d a b c
Farm number 1
2
3
4
5
6
7
8
9
929 143 9200 40 14 64
620 195 10,300 40 16 61
864 205 10,200 40 15 69
1020 197 10,400 60 16 70
497 140 9950 40 16 69
1380 123 10,200 50 15 79
1400 223 10,400 40 12 62
740 179 9800 50 15 69
1080 235 9600 50 15 60
Voluntary waiting period. 21d pregnancy rate. DIM corresponding to the first day of the TAI protocol.
2.4. Bulls and semen processing procedures Sires (n ¼ 10) producing the semen for this study were predominantly genomically selected Holstein-Friesian bulls. Sires were not randomly allocated to the farms due to individual breeding objectives on the farms. Ejaculates for producing liquid and frozen semen were collected using an artificial vagina per standard operating procedure twice a week (Monday and Wednesday) between 0600 and 0800 h. For the study only ejaculates from Wednesday were considered for the use of liquid semen. Ejaculates that met the required quality standards (i.e., >70% progressive motility; > 1.5 ml volume; > 500 106 sperm) were used for semen production. All ejaculates failing to meet these criteria were excluded from the study. The amount of liquid semen produced from each ejaculate was based on the requested amount of straws for that day. The remaining semen was used for the production of frozen semen. Frozen semen was diluted using OptiXcel liquid semen extender (IMV Technologies, Normandy, France) to 20 106 sperm/straw. Liquid semen was diluted using Caprogen liquid semen extender [8] to 10 106 sperm/straw. The 0.25 mL straws were filled with semen, sealed, and dispatched to technicians in the evening of the day of collection. During transport the straws were placed in a waterproof, insulated container, and submerged in water with a temperature of 10e15 C at the beginning. Frozen semen was transported to the farms and stored using containers with liquid nitrogen. Cows inseminated using the Ovsynch-56 protocol were
inseminated with liquid semen approximately 48 h after semen collection. Cows inseminated using the Cosynch-56 protocol were inseminated using liquid semen approximately 32 h after semen collection. 2.5. Statistical analyses Breeding information (i.e., cow ID, calving date, lactation number, breeding date, sire ID) and results of pregnancy diagnosis were obtained from the on farm computer system, exported to Excel (Office 2010, Microsoft Deutschland Ltd., Munich, Germany) spreadsheets and analyzed using SPSS for Windows (version 22.0, SPSS Inc., IBM, Ehningen, Germany). The analysis of P/AI at first TAI was performed by logistic regression using the GENLINMIXED procedure of SPSS. Cow was the experimental unit. Herd was considered as a random effect. According to the model-building strategies described by Dohoo et al. [22] each parameter considered for the mixed model should be separately analyzed in an univariate model, including the parameter as a fixed factor (i.e., categorical parameter) or covariate (i.e., continuous parameter). Only parameters resulting in univariate models with P 0.2 should be included in the final mixed model. Selection of the model that best fit the data was performed by testing each effect separately in an univariate model and finding the model with the lowest value for the Akaike information criterion (AIC) using a backward elimination procedure that removed all variables with P > 0.10 from the model. We used 2 different models in order to account for experiment 1 (Cosynch-56 and Ovsynch-56) and 2 (Ovsynch-56 only). 2.5.1. Experiment 1 The initial model contained the following explanatory variables as fixed effects: semen preservation method (frozen vs. liquid semen), TAI protocol (Ovsnych-56 vs. Cosynch-56), parity (primiparous vs. multiparous), sire (sire 1 to 10), DIM at first TAI (continuous). Regardless of the significance level type of semen and TAI protocol was forced to remain in the model.
Fig. 1. Experimental design. Lactating dairy cows were assigned randomly to 1 of 2 protocols for timed artificial insemination (TAI) according to the last digit of the ear tag number (odd or even). All enrolled cows in study experiment 1 received 100 mg of GnRH at GnRH1, or GnRH2 and 500 mg of PGF2a 7 d after GnRH1. Insemination was either performed concurrently with GnRH2 (Cosynch-56) or 12e16 h after GnRH2 (Ovsynch-56). In experiment 2, insemination was performed only 12e16 h after GnRH2 (Ovsynch-56). Semen preservation method (liquid vs. frozen) used for TAI changed every other week on each farm in experiment 1 and 2.
2.5.2. Experiment 2 In experiment 2, cows were only inseminated using the Ovsynch-56. Therefore, TAI protocol was not included in the model. The initial model contained the following explanatory variables as fixed effects: semen preservation method (frozen vs. liquid semen), parity (primiparous vs. multiparous), sire (sire 1 to 10), DIM at first TAI (continuous). Regardless of the significance level type of semen was forced to remain in the model. The adjusted P/AI values provided by SPSS are given throughout the text. The unadjusted values and actual experimental units (number pregnant divided by number inseminated) are also provided in Table 2.
24
S. Borchardt et al. / Theriogenology 107 (2018) 21e26
Table 2 Comparison of pregnancy per AI (P/AI) between cows inseminated with frozen semen or liquid semen and synchronized with Ovsynch-56 or Cosynch-56 for first timed artificial insemination in experiment 1 and 2. Parameter
Cosynch-56
Ovsynch-56
Frozen semen
Liquid semen
Frozen semen
Liquid semen
20.0x 19.5 60/307
27.5y 25.1 85/339
28.6 25.9 74/285
32.3 30.1 100/332
e e e
e e e
25.5 21.4 38/177
26.5 25.0 50/200
19.5 60/307
25.1 85/339
24.2 112/462
28.2 150/532
a
Experiment 1 Least square estimateb P/AI (%) n/n Experiment 2c Least square estimateb P/AI (%) n/n Overall P/AI (%) n/n x,y
Means within the same row with different superscripts differ (P < 0.05). In experiment 1 cows were either inseminated with liquid semen or frozen semen using either a Cosynch-56 or an Ovsynch-56. b Results obtained from the GENLINMIXED procedure. c In experiment 2 cows were either inseminated with liquid semen or frozen semen using an Ovsynch-56. a
3. Results Overall, 1724 AI were performed during this study. Eighty-four cows, however, were eliminated from the trial before pregnancy diagnosis because they were sold or died. Therefore, 1640 inseminations were used for statistical analyses.
injection (Ovsynch-56). In addition, parity affected P/AI with primiparous having a greater P/AI than multiparous cows (34.8% vs. 20.2%; P ¼ 0.001). There was no interaction of parity and semen type (P ¼ 0.872) or parity and TAI protocol (P ¼ 0.168). Sire had no effect on P/AI (P ¼ 0.899). 3.2. Experiment 2
3.1. Experiment 1 In experiment 1, the information of 1263 inseminations was available. There was no difference in DIM at first TAI between cows inseminated with frozen or liquid semen (P ¼ 0.604). The number of primiparous cows inseminated with frozen or liquid semen did not differ (P ¼ 0.700). The effect of semen preservation was conditional on TAI protocol. The results are summarized in Table 2 and Fig. 2. Cows inseminated with liquid semen concurrently with GnRH2 (Cosynch-56) achieved greater P/AI than cows inseminated with frozen semen using the same synchronization protocol (20.0% vs. 27.5%; P ¼ 0.032). There was no effect of semen preservation method (liquid semen 32.3% vs. frozen semen 28.6%; P ¼ 0.330) when cows were inseminated approximately 16 h after GnRH2
40
Pregnancy per AI, %
35 30 25 20 15 10 5 0 Frozen semen Liquid semen Frozen semen Liquid semen (n = 307) (n = 339) (n = 285) (n = 332) Cosynch-56
Ovsynch-56
Fig. 2. Comparison of pregnancy per AI between cows inseminated with frozen semen or liquid semen and synchronized either with Ovsynch-56 or Cosynch-56 for the first timed artificial insemination in experiment 1 using least square estimates (mean ± SEM) from the GENLINMIXED procedure.
In experiment 2, the information of 377 inseminations was available. There was no difference in DIM at first TAI between cows inseminated with frozen or liquid semen (P ¼ 0.532). The number of primiparous cows inseminated was greater (P ¼ 0.010) for cows inseminated with liquid semen (71/200) compared with cows inseminated with frozen semen (41/177). There was no effect of semen preservation method on P/AI (P ¼ 0.846). Parity affected P/AI with primiparous having a greater P/AI than multiparous cows (37.0% vs. 17.3%; P ¼ 0.001). There was no interaction of parity and semen type (P ¼ 0.929). Results for experiment 2 are summarized in Table 2. 4. Discussion This is the first randomized, controlled study to directly compare liquid semen with frozen semen in lactating dairy cows using timed AI protocols. Liquid semen achieved greater P/AI in a TAI protocol with a long time interval (~24 h) between insemination and ovulation (Cosynch-56) compared with frozen semen indicating that liquid semen might have a longer viability in the reproductive tract. There was no effect of semen preservation method on P/AI in a TAI protocol with a time interval from insemination to expected ovulation of 12e16 h (Ovsynch-56). While there is some evidence in beef cows indicating superior fertility of liquid semen [7,20,23], there is a dearth of published information on the fertility of liquid semen compared to frozen semen in dairy cows. Liquid semen processing yields more doses per ejaculate, thereby, facilitating the utilization of genetically superior sires. This is particularly beneficial for young genomically selected sires as these sires are in high demand but produce lower semen volumes in comparison to mature bulls [24]. In the absence of an improvement in P/AI of liquid semen over conventional frozen semen and with sufficient semen availability, there is little incentive for the dairy managers to use this product. For the AI companies a product with a limited shelf-life requiring a more sophisticated logistic is of limited value unless there is a higher
S. Borchardt et al. / Theriogenology 107 (2018) 21e26
conception risk. A recent study from Ireland evaluated the effect of 3 different semen diluents on P/AI using liquid semen in grazing dairy cows [25]. Overall, there was a decline in fertility for liquid semen, as measured by calving rate, with prolonged interval from semen collection until insemination. A reduced dose of liquid semen (5 106 sperm/straw) diluted in Caprogen or INRA96 resulted, however, in a similar fertility compared with frozen semen (15 106 sperm/straw). Our results indicated a superior fertility of liquid semen compared with frozen semen using only timed AI in high producing dairy cows in a confinement system. Results from these 2 studies can hardly be compared as they differ in sperm dose per straw for liquid and frozen semen, semen diluents, housing of cows, and method of insemination (i.e., AI after estrous detection vs. timed AI). Using liquid semen in timed AI protocols in conjunction with reduced sperm concentration and rapid overnight semen delivery may provide an opportunity to enhance utilization efficiency of dairy sires that are in high demand and short supply. Liquid semen minimizes time and labor necessary on the day of breeding because thawing of semen is not required. The freezethaw process can be an opportunity for human error with a negative impact on semen quality. Compared with frozen semen, the positive effect of liquid semen on P/AI was mainly attributed to the poor performance of frozen semen in the Cosynch-56 rather than the Ovsynch-56. Although there was a numerical increase of 4% points in P/AI in favor of the liquid semen considering the Ovsynch-56 the power (41.4%) of this study was not enough to detect a significant difference. Among other factors, success of timed AI programs depends on proper timing of insemination relative to ovulation [11]. For the standard Ovsynch program, with 7 days between the initial GnRH and PGF2a injection, maximum pregnancy risk in dairy cows can be achieved by administering the final GnRH 56 h after PGF2a and performing AI 16 h later [12]. The timing of AI, relative to the expected time of ovulation, is important for P/AI performing either AI after estrous detection [13] or timed AI [15]. The reduction in P/AI following Cosynch-56 is physiologically plausible based on the limited life span of sperm and the probable reduction in fertilization rate after a prolonged time from placing frozen thawed semen in the reproductive tract until ovulation [14]. This is in agreement with our results and supported by other studies [11,12,15] using frozen semen as well. The improvement in fertility, as measured by P/AI, by using Ovsynch-56 compared with Cosynch, is likely to offset an increase in labor costs for implementation of this protocol [15]. While this is applicable to frozen semen, our results indicate that in a Cosynch-56 using liquid semen dairy managers can save labor costs without affecting P/AI. In our study, primiparous cows had greater P/AI compared with multiparous cows irrespective of TAI protocol or semen preservation method. There are conflicting reports on the effect of parity on P/AI with the majority of studies showing a greater conception risk in primiparous cows [26e29] but some others showing no effect of parity [30e32]. This difference might be due to a parity specific response during the synchronization protocol [33]. In primiparous cows, a major risk factor for poor reproductive performance in a TAI protocol is the lack of a functional corpus luteum at the first GnRH injection. Therefore, primiparous cows benefit more from presynchronization using GnRH as they have a greater risk for anovulation [34,35]. Recent research also indicates that lacking luteolytic response in multiparous cows to a single prostaglandin administration in a 7 day Ovsynch protocol has a detrimental effect on P/AI [36]. For multiparous cows presynchronized with a Double-Ovsynch protocol an additional prostaglandin injection on d 8 of the breeding Ovsynch led to exceptional P/AI [32,36]. Besides parity-specific differences
25
related to successful synchronization in TAI protocols other contributors might be responsible for reduced fertility in multiparous cows such as the greater uterine size [37], greater BCS loss in early lactation [38], or a greater disease incidence in the transition period which was associated with reduced fertility later in lactation [39]. This study was initially conducted in a 2 2 factorial design to compare liquid and frozen semen using either an Ovsynch-56 or a Cosynch-56 protocol for the first TAI. After 5 months it became obvious that the Cosynch-56 was clearly inferior while using frozen semen. Therefore, we decided to terminate the Cosynch-56 to prevent economic disadvantages from the participating farms as reproductive performance is considered to be a major driver of overall farm profitability [40]. The premature termination of one study component was a compromise between the original study design and ethical considerations. We adapted to this change the experimental design by considering the 2 time periods (time period 1 Cosynch-56 and Ovsynch-56 and 2 Ovsynch-56 only) as 2 different experiments. A source of potential bias was the weekly change of semen used. This was done to improve overall compliance to a given protocol. We assumed that most farms would not be able to handle two different TAI protocols and two different semen handling methods concurrently without an increasing failure rate. Random allocation of cows into the timed AI protocol was based on the last digit of their unique 10-digit animal identification number. Ear tags were attached at birth of the calves in a consecutive order without knowledge of this study being implemented years later. Other studies randomizing cows based on their animal identification number had not been performed on the participating farms. Veterinarians conducting pregnancy diagnoses were not blinded to the study procedures and the allocation of cows to the protocols. The outcome, however, was of binary nature (i.e., pregnancy or non-pregnancy) and the veterinarians had no interest to make false-positive or false negative diagnoses. Therefore, we are comfortably sure that the results were not biased by the investigator. 5. Conclusions The results of this study provide evidence that liquid semen achieved greater P/AI in a Cosynch-56 protocol that is expected to have a longer interval between insemination and ovulation compared with frozen semen indicating that liquid semen might have a longer viability in the reproductive tract. Further research is warranted to evaluate the performance of liquid semen in cows inseminated after estrous detection with different intervals from AI to ovulation. Conflict of interest statement Neither of the authors of this paper has a financial or personal relationship with other people or organisations that could inappropriately influence or bias the content of the paper. Acknowledgements We are thankful to Veyx Pharma GmbH for providing the PGF2a and GnRH and the scientific collaboration. The authors are grateful to the owners and employees of the collaborating dairy farms for granting us access to their facilities, cows, and herd records and their wonderful support. Lisa Wolf was sponsored in part by Tiergyn e.V. Berlin.
26
S. Borchardt et al. / Theriogenology 107 (2018) 21e26
References [1] Stevenson JS. Impact of reproductive technologies on dairy food production in the dairy industry. Adv Exp Med Biol 2014;752:115e29. [2] NAHMS (national animal health monitoring service) reproductive practices on U.S. Dairy operations. 2007. [3] Gravance CG, Vishwanath R, Pitt C, Garner DL, Casey PJ. Effects of cryopreservation on bull sperm head morphometry. J Androl 1998;19:704e9. [4] Yoshida M. Conservation of sperms: current status and new trends. Anim Reprod Sci 2000;60e61:349e55. [5] Al Naib A, Ward F, Kelly AK, Wade M, Marti JI, Lonergan P. Effect of duration of storage at ambient temperature on fertilizing ability and mucus penetration ability of fresh bovine sperm. Theriogenology 2011;76:1070e5. [6] Xu ZZ. Application of liquid semen technology improves conception rate of sex-sorted semen in lactating dairy cows. J Dairy Sci 2014;97:7298e304. [7] Borges-Silva JC, Silva MR, Marinho DB, Nogueira E, Sampaio DC, Oliveira LO, et al. Cooled semen for fixed-time artificial insemination in beef cattle. Reprod Fertil Dev 2015;28:1004e8. [8] Vishwanath R, Shannon P. Storage of bovine semen in liquid and frozen state. Anim Reprod Sci 2000;62:23e53. [9] Thibier M, Wagner HG. World statistics for artificial insemination in cattle. 14th International Congress on Animal Reproduction. 2000. p. 76. [10] Caraviello DZ, Weigel KA, Fricke PM, Wiltbank MC, Florent MJ, Cook NB, et al. Survey of management practices on reproductive performance of dairy cattle on large US commercial farms. J dairy Sci 2006;89:4723e35. [11] Stevenson JS. Synchronization and artificial insemination strategies in dairy herds. Vet Clin North Am Food Anim Pract 2016;32:349e64. [12] Pursley JR, Silcox RW, Wiltbank MC. Effect of time of artificial insemination on pregnancy rates, calving rates, pregnancy loss, and gender ratio after synchronization of ovulation in lactating dairy cows. J Dairy Sci 1998;81: 2139e44. [13] Dransfield MB, Nebel RL, Pearson RE, Warnick LD. Timing of insemination for dairy cows identified in estrus by a radiotelemetric estrus detection system. J Dairy Sci 1998;81:1874e82. [14] Dalton JC, Nadir S, Bame JH, Noftsinger M, Nebel RL, Saacke RG. Effect of time of insemination on number of accessory sperm, fertilization rate, and embryo quality in nonlactating dairy cattle. J Dairy Sci 2001;84:2413e8. [15] Brusveen DJ, Cunha AP, Silva CD, Cunha PM, Sterry RA, Silva EP, et al. Altering the time of the second gonadotropin-releasing hormone injection and artificial insemination (AI) during Ovsynch affects pregnancies per AI in lactating dairy cows. J Dairy Sci 2008;91:1044e52. [16] Trimberger GW. Conception rate in dairy cattle by artificial insemination at various intervals before and after ovulation. J Dairy Sci 1944;27:659e60. [17] Portaluppi MA, Stevenson JS. Pregnancy rates in lactating dairy cows after presynchronization of estrous cycles and variations of the Ovsynch protocol. J Dairy Sci 2005;88:914e21. [18] Geary TW, Whittier JC. Effect of a timed insemination following synchronization of ovulation using the Ovsynch or Co-Synch protocol in beef cows. Prof Anim Sci 1998;14:217e20. [19] Curry MR. Cryopreservation of semen from domestic livestock. Rev Reprod 2000;5:46e52. [20] Bucher A, Kasimanickam R, Hall JB, Dejarnette JM, Whittier WD, Kahn W, et al. Fixed-time AI pregnancy rate following insemination with frozen-thawed or fresh-extended semen in progesterone supplemented CO-Synch protocol in beef cows. Theriogenology 2009;71:1180e5. [21] Nutrient requirements of dairy cattle. Washington, DC: Nat. Acad. Sci.; 2001. [22] Dohoo IR, Martin SW, Stryhn H. Veterinary epidemiologic research (2nd). Charlottetown, PEI, Canada: University of Prince Edward Island; 2009. [23] Papa PM, Maziero RD, Guasti PN, Junqueira CR, Freitas-Dell’Aqua CP, Papa FO, et al. Effect of glycerol on the viability and fertility of cooled bovine semen.
Theriogenology 2015;83:107e13. [24] Brito LF, Silva AE, Rodrigues LH, Vieira FV, Deragon LA, Kastelic JP. Effect of age and genetic group on characteristics of the scrotum, testes and testicular vascular cones, and on sperm production and semen quality in AI bulls in Brazil. Theriogenology 2002;58:1175e86. [25] Murphy EM, Murphy C, O'Meara C, Dunne G, Eivers B, Lonergan P, et al. A comparison of semen diluents on the in vitro and in vivo fertility of liquid bull semen. J Dairy Sci 2017;100:1541e54. [26] Tenhagen BA, Surholt R, Wittke M, Vogel C, Drillich M, Heuwieser W. Use of Ovsynch in dairy herds - differences between primiparous and multiparous cows. Anim Reprod Sci 2004;81:1e11. [27] Galvao KN, Sa Filho MF, Santos JE. Reducing the interval from presynchronization to initiation of timed artificial insemination improves fertility in dairy cows. J Dairy Sci 2007;90:4212e8. [28] Carvalho PD, Fuenzalida MJ, Ricci A, Souza AH, Barletta RV, Wiltbank MC, et al. Modifications to Ovsynch improve fertility during resynchronization: Evaluation of presynchronization with gonadotropin-releasing hormone 6 d before initiation of Ovsynch and addition of a second prostaglandin F2alpha treatment. J Dairy Sci 2015;98:8741e52. [29] Fuenzalida MJ, Fricke PM, Ruegg PL. The association between occurrence and severity of subclinical and clinical mastitis on pregnancies per artificial insemination at first service of Holstein cows. J Dairy Sci 2015;98:3791e805. [30] Giordano JO, Wiltbank MC, Guenther JN, Ares MS, Lopes Jr G, Herlihy MM, et al. Effect of presynchronization with human chorionic gonadotropin or gonadotropin-releasing hormone 7 days before resynchronization of ovulation on fertility in lactating dairy cows. J Dairy Sci 2012;95:5612e25. [31] Carvalho PD, Guenther JN, Fuenzalida MJ, Amundson MC, Wiltbank MC, Fricke PM. Presynchronization using a modified Ovsynch protocol or a single gonadotropin-releasing hormone injection 7 d before an Ovsynch-56 protocol for submission of lactating dairy cows to first timed artificial insemination. J Dairy Sci 2014;97:6305e15. [32] Carvalho PD, Wiltbank MC, Fricke PM. Manipulation of progesterone to increase ovulatory response to the first GnRH treatment of an Ovsynch protocol in lactating dairy cows receiving first timed artificial insemination. J Dairy Sci 2015;98:8800e13. [33] Borchardt S, Haimerl P, Pohl A, Heuwieser W. Evaluation of prostaglandin F2alpha versus prostaglandin F2alpha plus gonadotropin-releasing hormone as Presynch methods preceding an Ovsynch in lactating dairy cows: a metaanalysis. J Dairy Sci 2017;100:4065e77. [34] Souza AH, Ayres H, Ferreira RM, Wiltbank MC. A new presynchronization system (Double-Ovsynch) increases fertility at first postpartum timed AI in lactating dairy cows. Theriogenology 2008;70:208e15. [35] Herlihy MM, Giordano JO, Souza AH, Ayres H, Ferreira RM, Keskin A, et al. Presynchronization with Double-Ovsynch improves fertility at first postpartum artificial insemination in lactating dairy cows. J Dairy Sci 2012;95: 7003e14. [36] Wiltbank MC, Baez GM, Cochrane F, Barletta RV, Trayford CR, Joseph RT. Effect of a second treatment with prostaglandin F2alpha during the Ovsynch protocol on luteolysis and pregnancy in dairy cows. J Dairy Sci 2015;98:8644e54. [37] Baez GM, Barletta RV, Guenther JN, Gaska JM, Wiltbank MC. Effect of uterine size on fertility of lactating dairy cows. Theriogenology 2016;85:1357e66. [38] Carvalho PD, Souza AH, Amundson MC, Hackbart KS, Fuenzalida MJ, Herlihy MM, et al. Relationships between fertility and postpartum changes in body condition and body weight in lactating dairy cows. J Dairy Sci 2014;97: 3666e83. [39] Santos JE, Bisinotto RS, Ribeiro ES, Lima FS, Greco LF, Staples CR, et al. Applying nutrition and physiology to improve reproduction in dairy cattle. Soc Reprod Fertil Suppl 2010;67:387e403. [40] Cabrera VE. Economics of fertility in high-yielding dairy cows on confined TMR systems. Animal 2014;8(Suppl. 1):211e21.