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Animal Reproduction Science 143 (2013) 19– 23

Contents lists available at ScienceDirect

Animal Reproduction Science

jou rn al hom epage : w ww.elsev ier .com/ locate /an i r eprosc i

se of sex-sorted sperm in lactating dairy cows upon estrusetection or following timed artificial insemination

.F. Sá Filho ∗, M.F. Mendanha, R.V. Sala, F.J. Carvalho,.H.C. Guimarães, P.S. Baruselli ∗

epartamento de Reproduc ão Animal, FMVZ-USP, Rua Prof. Orlando Marques de Paiva 87, CEP 05508-000, São Paulo, SP, Brazil

a r t i c l e i n f o

rticle history:eceived 9 August 2013eceived in revised form 21 October 2013ccepted 27 October 2013vailable online 7 November 2013

eywords:eproductionairy cowseproductive performanceynchronization of ovulation

a b s t r a c t

The present study evaluated the use of sex-sorted sperm upon estrus detection (ED) or fol-lowing timed artificial insemination (TAI) in lactating dairy cows. Additionally, the effectof the presence of a corpus luteum (CL) at the beginning of the TAI protocol was verified.Cows (539 crossbred Gir × Holstein and 87 Holstein) were classified according to the pres-ence or absence of CL by ultrasonography exam. Cows with a CL were randomly assignedinto one of two groups (CL-ED/AI or CL-TAI), and cows without a CL (NoCL-TAI) receivedTAI. Cows from the CL-ED/AI group received 500 mg of cloprostenol intramuscularly andwere inseminated 12 h after ED in the following five days. Cows from the TAI groups (CL orNoCL) received TAI. Cows receiving CL-ED/AI had a lower (P < 0.0001) service rate (45.1%,101/224) than TAI groups (CL-TAI = 94.2%, 180/191 and NoCL-TAI = 97.2%, 205/211). How-ever, cows receiving AI upon ED (CL-ED/AI = 31.7%, 32/101) presented higher (P = 0.03)pregnancy per AI (P/AI) than cows bred following TAI (CL-TAI = 19.4%, 35/180 and NoCL-TAI = 23.9%, 49/205). Despite the lower P/AI, cows receiving TAI presented greater (P = 0.07)proportion of pregnant cows at the end of the reproductive program (CL-TAI = 18.3%, 35/191

and NoCL-TAI = 23.2%, 49/211) than those inseminated upon ED (14.3%, 32/224). There wasno effect (P = 0.45) of the presence of a CL at the beginning of the synchronization protocolon P/AI. Thus, the use of TAI programs, regardless of the presence of CL in the beginningof the synchronization protocol, increases the service and pregnancy rates but reduces theP/AI when compared to the use of sex-sorted sperm upon ED.

. Introduction

Sex-sorting of sperm cells by flow cytometry is an estab-ished method that has been commercially used in dairyattle production (Seidel, 2007; Garner and Seidel, 2008;ath et al., 2013). Sex-sorted sperm is an important tech-

ology for the dairy industry because it creates a greaterupply of replacement heifers and hastens the genetic gainrogress (De Vries et al., 2008; Chebel et al., 2010). Despite

∗ Corresponding authors. Tel.: +55 11 3091 7674;ax: +55 11 3091 7412.

E-mail addresses: mfsvt@yahoo.com, manoelsa@usp.br (M.F. Sá Filho),arusell@usp.br (P.S. Baruselli).

378-4320/$ – see front matter © 2013 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.anireprosci.2013.10.014

© 2013 Elsevier B.V. All rights reserved.

the advances in sex-sorting of sperm using flow cytome-try, lower pregnancy per AI (P/AI) has been achieved thanwhen using conventional semen (DeJarnette et al., 2009,2010, 2011; Sales et al., 2011; Sá Filho et al., 2012).

The P/AI in cattle could be influenced by several factorswhen the sex-sorted sperm is applied (Seidel and Schenk,2008; DeJarnette et al., 2009; Frijters et al., 2009; Schenket al., 2009; Sá Filho et al., 2010). The reduced lifespan in theuterus, reduced number of sorted sperm per straw and bull-related fertility effect could be suggested to be the main

factors determining the reduced fertility after the use ofsex-sorted sperm in cattle (Maxwell et al., 2004; Frijterset al., 2009; Underwood et al., 2009; DeJarnette et al., 2010,2011; Underwood et al., 2010; Gosálvez et al., 2011). The

producti

20 M.F. Sá Filho et al. / Animal Re

reduced lifespan of the sex-sorted sperm in the femalereproductive tract, due to mitochondria modification andDNA fragmentation, could alter the optimum interval toperform the AI relative to ovulation (Maxwell et al., 2004;Sá Filho et al., 2010; Gosálvez et al., 2011; Sales et al., 2011;Rath et al., 2013). Furthermore, different fertility levelsamong bulls have been considered paramount when sex-sorted semen is utilized in commercial AI programs (Seideland Schenk, 2008; Frijters et al., 2009; DeJarnette et al.,2011; Sales et al., 2011).

Currently, the major commercial recommendation forthe use of sex-sorted sperm has been in dairy heifers afterestrus detection (ED), especially when trying to achievethe traditionally high P/AI (DeJarnette et al., 2009; Normanet al., 2010; Healy et al., 2013). Likewise, others haveincreased the use of sex-sorted sperm in inseminating first-service lactating dairy cows upon ED (Norman et al., 2010).However, although these strategies can produce accept-able results in terms of P/AI, they are incompatible withnewer trends in reproductive management used in moderndairy operations, e.g., extensive TAI synchronization pro-grams (Caraviello et al., 2006). Currently, the systematicincorporation of TAI programs in the routine reproductivemanagements of farms has quickly replaced traditionallybreeding based on ED. Therefore, it could be important toevaluate the reproductive performance of lactating dairycows inseminated using sex-sorted sperm upon traditionalED compared to those following TAI breeding program.

The main focus of the present study was to evaluatethe use of sex-sorted sperm in lactating dairy cows uponED or following TAI. An additional objective was to com-pare the reproductive outcomes (i.e., service rate, P/AI andpregnancy rate) of these two different breeding programs.Lastly, the effect of the presence of a CL at the beginning ofthe TAI protocol of lactating dairy cows inseminated withsex-sorted sperm was determined. The hypothesis inves-tigated was that dairy cows inseminated with sex-sortedsperm after TAI synchronization programs present greaterservice and pregnancy rates and similar P/AI to cows sub-jected to AI upon ED. Additionally, the presence of a CL atthe onset of synchronization of ovulation protocol does notaffect the P/AI.

2. Materials and methods

2.1. Farm and animals

This experiment was conducted in a commercial dairyfarm in southwest Brazil (22◦34′48′′ S, 44◦41′49′′W) fromAugust of 2010 to December of 2012. Lactating cows [539crossbred Gir × Holstein (1/2 Holstein; n = 71, 3/4 Holstein;n = 402 and 7/8 Holstein; n = 66) and 87 Holstein] housedin dry lot corral (n = 9 corrals) were used. Lactating cows(210 primiparous and 416 multiparous) were enrolled toreceive the first (n = 203), second (n = 213) and third (n = 70)postpartum service. Cows presented 77.0 ± 1.7 days in milkand an average of milk production of 23.8 ± 0.4 L/day at the

beginning of the reproductive management. Body condi-tion scores were evaluated on the first day of the breedingprogram using a 1–5 scale (Ferguson et al., 1994). Cowswere milked twice daily and fed with silage as forage and a

on Science 143 (2013) 19– 23

corn, distilled grain residues and soybean meal-based con-centrate, which exceeded the nutritional requirements oflactating dairy cows (NRC, 2001).

2.2. Reproductive management and experimental design

After the voluntary waiting period of 45 days, cowswere enrolled in the study every 14 days, totalizing 25managements (i.e., breeding groups). During each herdmanagement, cows had their ovaries examined for thepresence of a corpus luteum (CL) by ultrasound. Non-pregnant cows presenting CL were randomly assignedinto one of two treatment groups and subjected to AIupon estrus detection (CL-ED/AI; n = 224) or TAI (CL-TAI;n = 191). Nevertheless, non-pregnant cows not presentinga CL at the ultrasound examination were synchronizedto receive TAI (NoCL-TAI; n = 211). On a random day ofthe estrous cycle, cows from the CL-ED/AI group received500 mg of cloprostenol (Ciosin®, MSD Animal Health,Brazil) intramuscularly and were observed twice dailyfor estrus for the following five days. Immediately afterPGF2� administration, cows received an adhesive ED aid(Estrotect®, IVP, Spring Valley, WI, United States), whichwas placed between the hips and the tail head. Estruswas determined by the detection of the cow standingheat or by the activation of the ED aid. Cows were arti-ficially inseminated 12 h after ED. Cows from the TAIgroups (CL-TAI and NoCL-TAI) had their ovulation syn-chronized to receive TAI. The synchronization of ovulationprotocol consisted of the insertion of a new or a pre-viously used (8 days of use) intravaginal progesterone(P4) device (DIB®, MSD Animal Health, Brazil) associ-ated with 2 mg of estradiol benzoate (EB; Gonadiol®, MSDAnimal Health, Brazil). Eight days later, the P4 devicewas removed, and 500 mg of cloprostenol (Ciosin®, MSDAnimal Health, Brazil) was intramuscularly administered.After the initial use, the P4 devices were individuallywashed with water and then soaked in a solution of chlo-ride alkyl dimethyl benzyl ammonium (CB 30®, OurofinoAgronegocio, São Paulo, Brazil) for approximately 10 min.Thereafter, the P4 devices were dried using brown paper,placed inside aluminum bags and stored at room tempera-ture until use. Cows received 1 mg of EB intramuscularly24 h after the removal of the P4 device, and insemina-tion was timed for 36 h later (Sales et al., 2011) (Fig. 1).Frozen-thawed sex-sorted sperm (2.1 × 106 sperm) from11 Holstein bulls was evenly distributed among thethree groups.

2.3. Procedures for sexing and processing sperm

The sexing of sperm at 85–90% accuracy was accom-plished with a MoFlo® SX sperm sorter (DakoCytomation,Sexing Technologies, Navasota, TX, USA) operated at35 psi and approximately 40,000 events/s, resulting insort rates of 5000–8000 sperm/s. Sperm were stained at160 × 106 sperm/mL and sorted at 80 × 106 sperm/mL fol-

lowing filtering at unit gravity through a 50 CellTrics®

disposable filter (#04-0042-2317; Partec GmbH, Mun-ster, Germany). Sperm were stained with 112.5 M Hoechst33342 and interrogated with 150 mW laser intensity.

M.F. Sá Filho et al. / Animal Reproduction Science 143 (2013) 19– 23 21

PGF2 + Estrotect®

CL-ED/AI g roup (n= 224 cows )

2mg EB

24 h

TAI

PGF2

1mg EB

36 h

AI upon estrus detection

P4 device

2mg EB

24 h

TAI

PGF2

1mg EB

36 hP4 device

US

exam

With

CL

Without

CL

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NoCL-TAI group( n= 2 11 cows )

0Days 8 9 105

Fig. 1. Cows were enrolled depending on the presence of a corpus luteum (CL) by ultrasound (US) at the onset of the breeding program. Cows presentinga CL were randomly assigned into one of two groups and subjected to AI upon estrus detection (CL-ED/AI; n = 224) or timed artificial insemination (TAI;CL-TAI; n = 191). Nevertheless, all cows presenting an absence of a CL were synchronized to receive a TAI (NoCL-TAI; n = 211). Cows from the CL-ED/AI groupreceived 500 mg of cloprostenol (PGF ) intramuscularly plus adhesive estrus detection aid (Estrotect®) and were observed twice daily for estrus duringt gesterol eived 1r

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he following five days. Cows from TAI groups received an intravaginal proater, the P4 device was removed, and PGF2�was administered. Cows receceived timed insemination 36 hours later.

perm were frozen in a 20% egg yolk-Tris extender (6%lycerol) and packaged in 0.25-mL polyvinyl chloride straw2.1 × 106 sperm per straw).

.4. Ultrasound exams and pregnancy diagnosis

On every routine herd reproductive visit, cow ovariesere examined by transrectal ultrasonography (DP 2200ET, Mindray, Guangdong, China) to verify the presence of

CL. The pregnancy diagnoses were performed by ultra-ound 35 ± 7 days after AI. Detection of an embryonicesicle with a viable embryo (presence of a heartbeat) wassed as a positive indicator of pregnancy. Pregnancy perI (P/AI) was defined as the number of cows pregnantfter AI divided by the total number of females insemi-ated. The pregnancy rate was defined as the number ofows pregnant after AI divided by the total number of cowsnrolled in each experimental group (CL-ED/AI, CL-TAI oroCL-TAI).

.5. Statistical analysis

The binomial responses such as service rate, P/AI and

regnancy rate were evaluated using the GLIMMIX proce-ure from the SAS program (SAS Institute Inc., Cary, NC,SA). Orthogonal comparisons were performed to deter-ine the effects of the breeding strategy (PGF+ED versus

ne (P4) device associated with 2 mg of estradiol benzoate (EB). Eight days mg of EB intramuscularly 24 h (h) after the P4 device was removed and

CL-TAI and NoCL-TAI) and the effect of the presence ofCL at the beginning of the synchronization protocol (CL-TAI versus NoCL-TAI) on reproductive parameters. Theinitial model used to analyze all reproductive parame-ters included the fixed effects of treatment (CL-ED/AI,CL-TAI and NoCL-TAI), number of services (1 to 3), breed[crossbred Gir × Holstein (1/2 Holstein, 3/4 Holstein and7/8 Holstein) and Holstein], parity (multiparous or prim-iparous), BCS at the onset of the breeding program (1–5scale), and interactions between treatment and othercovariates. The breeding group within the corral wasincluded as a random effect. The initial model used toanalyze the P/AI also included the bull as random effect.Variables were removed through backward eliminationbased on the Wald statistics criterion when P > 0.20. Thefinal model for the service rate, P/AI and pregnancy ratesincluded only the variable treatment (CL-ED/AI, CL-TAI andNoCL-TAI).

3. Results

No effects of number of services (P = 0.94), parity(P = 0.37), breed (P = 0.92) and BCS at the onset of the

breeding program (P = 0.40) influencing the service ratewere observed. However, there was a significant effect(P < 0.0001) of the breeding strategy (ED vs. TAI) on theservice rate (Table 1).

22 M.F. Sá Filho et al. / Animal Reproduction Science 143 (2013) 19– 23

Table 1Reproductive parameters in lactating dairy cows subjected to different breeding programs for the use of sex-sorted sperm.

Treatmenta Contrastb – P valuesCL-ED/AI CL-TAI NoCL-TAI Breeding strategy Presence of CL

No. of cows 224 191 211 – –Service ratec (%) 45.1 (101/224) 94.2 (180/191) 97.2 (205/211) <0.0001 0.16Pregnancy per AI (%) 31.7 (32/101) 19.4 (35/180) 23.9 (49/205) 0.03 0.45Pregnancy rated (%) 14.3 (32/224) 18.3 (35/191) 23.2 (49/211) 0.07 0.28

a Cows presenting a corpus luteum (CL) on the first day of the breeding program were randomly assigned into one of two treatment groups and subjectedto AI upon estrus detection (CL-ED/AI) for five days after PGF2� treatment or timed artificial insemination (TAI; CL-TAI). Nevertheless, all cows without aCL were synchronized to receive a TAI (NoCL-TAI).

b Orthogonal comparisons were performed to determine the effects of breeding strategy (CL-ED/AI vs. CL-TAI plus NoCL-TAI) and the presence of a CL atI).

by the tdivided

the beginning of the synchronization protocol for TAI (CL-TAI vs. NoCL-TAc Service rate was defined as the number of cows inseminated divided

d Pregnancy rate was defined as the number of cows pregnant after AI

The P/AI did not alter depending on the num-ber of services [first = 24.1% (49/203); second = 24.4%(52/213) and third = 21.4% (15/70); P = 0.99), parity [primi-parous = 21.8% (37/170) and multiparous = 25.0% (79/316);P = 0.77], breed [1/2 Holstein = 24.0% (12/50), 3/4 Hol-stein = 25.4% (79/311), 7/8 Holstein = 23.2 (13/56) andHolstein = 17.4 (12/69); P = 0.36] 1/2 3/4 7/8 and BCS(2.5 = 11.8% (2/17) 2.75 = 17.3% (9/52), 3.0 = 23.3% (28/120);3.25 = 25.2% (28/111); 3.5 = 24.1% (33/137); 3.75 = 32.7%(16/49); P = 0.55). However, cows bred using sex-sortedsperm after ED presented greater P/AI than cows bred fol-lowing TAI (P = 0.03; Table 1). No difference was found(P = 0.45) in the P/AI among cows with or without a CL at thebeginning of the synchronization protocol for TAI (Table 1).There was a significant effect (P = 0.05) of the bull on theP/AI. Additionally, greater variation in fertility (i.e., P/AI)among bulls (ranged from 11.6 to 46.1%) was observed.

There was no influence of the number of service(P = 0.59), parity (P = 0.77), breed (P = 0.44) and BCS(P = 0.65) on the pregnancy rate. However, cows receivingTAI presented greater (P = 0.07) pregnancy rates than thoseinseminated upon ED (Table 1). No difference was found(P = 0.28) on the pregnancy rates depending on the pres-ence of a CL at the onset of the ovulation synchronizationprogram for TAI.

4. Discussion

This study evaluated practical strategies for the use ofsex-sorted sperm in lactating dairy cows. Dairy cows bredupon ED presented a lower service rate but higher P/AIusing sex-sorted sperm than cows receiving a TAI, partiallyvalidating the present hypothesis. However, dairy cowsreceiving a TAI using sex-sorted sperm presented a higherpregnancy rate than cows bred after the detection of estrus.Furthermore, there was no effect of the presence of a CLat the onset of the synchronization of ovulation programon the evaluated reproductive parameters, confirming thepresent hypothesis.

A lower service rate was achieved following the AIperformed based on ED after PGF2� treatment than cowssubjected to the TAI program, regardless of the presence

of a CL at the onset of the TAI synchronization protocol.The implementation of AI programs based on ED in lac-tating dairy herds is hampered mainly by ED failure, thelarge size of farms, the high number of animals per lot, and

otal number of females enrolled in the study.by the total number of cows enrolled.

labor costs. The problem of low ED has been reported byother groups working with lactating dairy cows (Cordobaand Fricke, 2001, 2002; Cavestany et al., 2007; Gutiérrezet al., 2009) and is considered a critical factor responsiblefor the poor reproductive efficiency of ED-based reproduc-tive programs (Senger, 1994). Nevertheless, TAI programsmay eliminate the need for ED and also provide a system-atic approach for the use of AI with either non-sex-sortedor sex-sorted sperm. Additionally, the TAI programs enablethe quick enrollment of cows without a CL in the breedingprograms. These animals would not respond to the estroussynchronization program using PGF2� and thus presentlower reproductive performance.

Regardless of the breeding strategy, the general P/AI oflactating dairy cows bred with sex-sorted sperm found inthe present study was 24% (116/486). A previous retrospec-tive report demonstrated that the use of sexed semen forthe AI of US Holstein cows (10.8 million AI) resulted in amean conception rate of 25% (Norman et al., 2010). A sim-ilar P/AI has been described in other scientific reports thatcompared the use of conventional or sex-sorted sperm inlactating dairy cows (Andersson et al., 2006; Schenk et al.,2009; DeJarnette et al., 2010). Furthermore, it is impor-tant to note that a large range in P/AI among individualsires (11.6–46.1%) was found in the present study. Theindividual-bull differences can notably affect pregnancyoutcomes when sex-sorted semen is used (DeJarnette et al.,2009, 2011; Sales et al., 2011). Thus, the pre-determinationof sire fertility is still a major concern when sex-sortedsperm is employed in commercial AI programs.

Cows inseminated after the TAI program in the presentstudy presented greater pregnancy rates than cows insem-inated upon ED. However, cows receiving a TAI withsex-sorted sperm, regardless of the presence of CL at thebeginning of the synchronization protocol, presented alower P/AI than those inseminated after ED. There is limitedscientific information regarding the impact of reproductivemanagement on the P/AI after the use of sex-sorted spermin lactating dairy cows. Moreover, the present results arecontrary to several reports using conventional semen inwhich the conception rate of lactating dairy cows receivingTAI was similar to that of cows receiving AI after a stand-

ing estrus (Pursley et al., 1995, 1997; Santos et al., 2009;Teixeira, 2010). However, the difference on the P/AI accord-ing to the breeding strategy found here is in agreementwith other studies that reported lower conception rates in

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ows bred following a TAI program compared to those cowseceiving AI after ED (Stevenson et al., 1999; Jobst et al.,000). The factors responsible for the variation in P/AI withAI are unknown at this moment but could consist of theariation in the ovarian follicular response of the individ-al cow after the synchronization of ovulation program orhe insemination-ovulation interval especially when sex-orted sperm is applied. This requires further studies using

greater number of animals.In conclusion, the use of TAI programs in lactating dairy

ows, regardless of the presence of a CL in the beginning ofhe synchronization protocol, increases the service rate, buteduces the P/AI when compared to the use of sex-sortedperm upon ED.

cknowledgements

The authors would like to thank the staff of the Fazendaao Miguel (Areias-SP) for allowing the use of their animalsnd facilities during this study. The authors also acknowl-dge the doctors Bruno M. Monteiro, Julia G. Soares andustavo G. Macedo for their participation in the project.his research was supported by Sexing Technologies BrasilSertaozinho-SP), MSD Animal Health (Sao Paulo-SP), IVP-rasil (Sao Paulo-SP) and CAPES.

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