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Theriogenology 81 (2014) 67–73

Contents lists ava

Theriogenology

journal homepage: www.ther io journal .com

40th Anniversary Special Issue

Bovine in vitro fertilization: In vitro oocyte maturation and spermcapacitation with heparin

John J. Parrish*

Department of Animal Sciences, University of Wisconsin, Madison, Wisconsin, USA

a r t i c l e i n f o

Article history:Received 12 July 2013Received in revised form 6 August 2013Accepted 7 August 2013

Keywords:IVFFertilizationHeparinCapacitationBovine

* Corresponding author. Tel.: þ1 608 263 4324; fE-mail address: parrish@ansci.wisc.edu.

0093-691X/$ – see front matter � 2014 Elsevier Inchttp://dx.doi.org/10.1016/j.theriogenology.2013.08.0

a b s t r a c t

As a result of research in the 1980s on in vitro maturation, sperm capacitation, and in vitrofertilization, the bovine is now one of the important models for development. Further, thecurrent production of bovine embryos in vitro rivals that of in vivo embryo production forcommercial applications. Researchers of today may be unaware of why decisions weremade in the procedures. This review addresses the state of the art at the time of the workby Parrish et al. (Bovine in vitro fertilization with frozen thawed semen. Theriogenology1986;25:591–600), and how later work would explain success or failure of competingprocedures. Important was the use of frozen semen and heparin capacitation, because thisallowed future researchers/practitioners to change sperm numbers and capacitationconditions to adjust for variations among bulls. The large numbers of citation of theoriginal work stand the testament of time in the repeatability and success of the pro-cedures. The work was done within the environment of the N.L. First laboratory and theunique interactions with a large number of talented graduate students, postdoctoral re-searchers, and technicians.

� 2014 Elsevier Inc. All rights reserved.

1. Introduction

The report of in vitro fertilization (IVF) of bovine oo-cytes with frozen thawed semen and using heparin [1] hasbeen important to most subsequent work with bovine IVFfor research or the commercial production of embryos.The purpose of the experiments was to demonstrate thatheparin was capable of increasing the ability of bovinesperm to fertilize bovine oocytes in vitro. The work wasbuilt on research by others in the First and Ax laboratoriesat the University of Wisconsin as well as Bracket et al. atthe University of Pennsylvania [2,3]. This review beginswith a discussion of the in vitro maturation procedures in1986 andwhy results of IVF were reported differently thanmost researchers would recognize today. A discussionof the status of IVF and sperm capacitation in 1986, andhow understanding heparin-induced capacitation now

ax: þ1 608 262 5157.

. All rights reserved.05

explains why other methods used to capacitate sperm inthe 1980s likely succeeded follows. The final section dealswith current impacts of heparin and IVF in the in vitroproduction of embryos for research and commercialtransfer. This review does not address culture conditionsfor embryo development, because this was not part of theoriginal publication [1].

2. In vitro maturation of oocytes

The oocyte maturation procedure used in Parrish et al.[1] and other publications associated with the First and Axlabs from 1983 to 1986 used a procedure with a Tyrode’sbase medium that was supplemented with fetal calf serumand a FSH preparation that had LH activity as described inBall et al. [4]. Although this succeeded in maturing oocytesin vitro to the stage at which oocytes were arrested atmetaphase II of meiosis, it still had deficiencies. An ovu-lated oocyte would be at this same stage when penetratedby a sperm in the oviduct, but would then be capable of

Fig. 1. In vitro–matured and fertilized bovine oocyte. The oocyte was one thefirst matured in vitro and fertilized with heparin-treated sperm in early 1984as described in Parrish et al. [1]. Two pronuclei (PN) are shown along withthe tail of the penetrating sperm (ST).

J.J. Parrish / Theriogenology 81 (2014) 67–7368

forming both paternal and maternal pronuclei. Paternalrefers to the sperm-derived pronculei and maternal to theoocyte-derived pronuclei. This was not true of the in vitrooocyte maturation method described. To fully describesuccessful penetration, fertilization was expressed aspenetration by sperm, two-pronuclear formation, and oo-cytes with evidence of penetration by only one sperm or amaternal pronuclei present. An example of one of the firstoocytes fertilized by heparin-treated sperm in early 1984 isshown in Figure 1. The maturation of bovine oocytes underthe conditions of Ball et al. [4] often resulted in reducedpaternal pronuclei formation. Maternal pronuclei seemedto form if the oocytes were activated by sperm penetration.It would be found later that estrogen was required and theTyrode’s-based medium needed to be changed to a morecomplete cell culture medium, namely, Medium 199 [2,5].In addition, the gonadotropins FSH and LH were now ob-tained from purified National Institute of Arthritis, Meta-bolism and Digestive Disease origin. These changes weresufficient for paternal pronuclear formation and supportedfull development [5,6,7] and the birth in 1986 of a calf fromin vitro–matured oocytes and IVF. Rarely is failure ofpaternal pronuclei noted anymore with in vitro–maturedbovine oocytes. The key was most likely the inclusion ofestrogen in the maturation medium. Many investigationsby others were ongoing at the time using different serumsupplements and co-culture of oocytes during maturationwith other cell types [2], but the basic method [5,6] isnow standard, with only modifications to source ofgonadotropins.

3. Capacitation of sperm

Once oocytes are matured, it is critical to expose thoseoocytes to sperm that have already been capacitated or are

undergoing capacitation. Capacitated sperm have under-gone biochemical modifications that allow them to acro-some react upon exposure to the zona pellucida, cumuluscells, or other substances associated with in vitro–maturedor ovulated oocytes [8,9,10,11]. In the mid 1980s, it was notalways clear how specific sperm procedures impactedsperm to enhance IVF in the bovine. Effects could have beenon capacitation, the acrosome reaction, or both. If spermwere capacitating during incubation with oocytes, it wasalso important to consider whether oocytes would agebefore sperm were capacitated and able to penetrate thezona pellucida. The source of spermdejaculated unfrozenor cyropreserveddis also critical. One unique aspect ofwork by the Parrish et al. [1] was the use of frozen-thawedsemen. However, using frozen-thawed semen results inmany more sperm dying over incubation than would beseen with unfrozen semen. Such dead sperm complicatethe interpretation of what is happening either before orduring incubation with oocytes. Most of the works wedescribe have used unfrozen semen for just this reason.

Bracket et al. in a series of reports [12,13,14] demon-strated that brief exposure of washed bovine semen to highionic strength media (HIS) induced sufficient capacitationfor sperm to fertilize in vivo–matured bovine oocytes. TheHIS medium was made by adding sufficient NaCl to theBracket-Oliphant medium (BO; Table 1) to achieve 380mosmols. The HIS and BO media had previously beenshown to induce capacitation of rabbit sperm by presum-ably displacing decapaciation factors from the surface ofsperm [15]. The results of treating either fresh or frozen-thawed bovine sperm with HIS treatment for IVF pro-duced only modest penetration of oocytes by sperm. It wasdifficult to replicate this work and many results may havebeen dependent on the use of semen from particular bulls.A further limitation may have been that sperm were notsufficiently capacitated.

A different approach for capacitating bovine semen wasalso being developed by Ax et al. [4,16], and related to thepossible role that follicular fluid and/or oviduct secretionsplay in capacitating or inducing the acrosome reaction insperm. Follicular fluid and oviduct secretions are rich inglycosaminoglycans (GAGs) [17] and sperm were able toundergo the acrosome reaction after exposure to thesecompounds [4,16]. However, Parrish et al. [18] were unableto demonstrate effects of the GAG, chondroitin sulfate A, inits ability to stimulate either acrosome reactions or fertil-ization frequencies. Heparin was found to stimulate boththe acrosome reaction and fertilization, but an interactionwith the presence of glucose in the media was noted.Confusion had arisen from the description of the Tyrode’smedium used by the previous studies. In the hamster,where themediumwas originally described, glucosewas inthe final formulation [19]. It was unclear whether glucosewas used in the Tyrode’s medium that was described foruse with bovine sperm and GAGs [4,16]. It was discoveredthat the Tyrode’s medium was made in two different lab-oratories, where people had different backgrounds thatinfluenced whether they included glucose or not in me-dium. It would later be found that glucose delayed capac-itation of bovine sperm by heparin [18]. Researchers shouldbe aware that simply referencing a media may not be

Table 1Common media used for capacitation and fertilization of bovine gametes.a

Component BOb Sp-TALPc Sp-TALP-Hd TL-HEPESe Fert-TALPf

NaCl (mmol/L) 112.00 100.00 87.00 114.00 114.00KCl (mmol/L) 4.02 3.10 3.10 3.10 3.20CaCl2 (mmol/L) 2.25 2.00 2.00 2.00 2.00NaH2PO4 (mmol/L) 0.83 0.30 0.30 0.30 0.30MgCl2 (mmol/L) 0.52 0.40 0.40 0.50 0.50NaHCO3

– (mmol/L) 37.00 25.00 10.00 2.00 25.00HEPES (mmol/L) d 10.00 40.00 10.00 d

Glucose (mmol/L) 13.90 0 0 0 0Pyruvic acid (mmol/L) 1.25 1.00 1.00 0.20 0.20Lactic acid (mmol/L) d 21.60 21.60 10.00 10.00BSA (mg/mL) 10 6 6 3 6Penicillamine (mmol/L) 0–20 d d d 20Hypotaurine (mmol/L) 0–10 d d d 10Epinephrine (mmol/L) 0–1 d d d 1SodiumMetabisulfite (mmol/L) ?g d d d 2

a Formulations were from several publications [3,20,21,36]. Most media utilize some type of antibiotic such as 50 mg/mL gentamycin.b Bracket and Oliphant medium [15].c Stands for sperm TALP (Tyrode’s medium base, albumin, lactate and pyruvate). Also known as bovine gamete medium 1 (BGM1) in Parrish lab publi-

cations. Must be incubated under 5% CO2 in air to maintain pH.d The H is for high amount of HEPES. This medium should be incubated in air and will support IVF under an air atmosphere if desired. This medium is also

known as BGM3 in Parrish lab publications.e Medium used to wash oocytes before IVF.f Fertilization TALP as described in Parrish et al. [1]. The medium must be kept under a 5% CO2 in air atmosphere to maintain pH.g ?, It is not known if sodiummetabisulfite was present but it is used to stabilize the penicillamine, hypotaurine, and epinephrine preparation and directly

improves IVF results.

J.J. Parrish / Theriogenology 81 (2014) 67–73 69

sufficient if slight changes have been made during devel-opment of the medium; precise formulations should belisted to avoid confusions. Compositions of various mediaused in oocyte handling, sperm preparation, and during IVFare listed in Table 1. The handling of oocytes or embryosbetween incubations is done in TL-HEPES that has reducedlevels of bicarbonate and HEPES added tomaintain pH in anair atmosphere. Incubation of unfrozen semen is done inSp-TALP, but requires gassing of media and tubes with 5%CO2 in air because the medium contains 25 mmol/L bicar-bonate and only 10 mmol/L HEPES [20]. An alternative isthe Sp-TALP-H, which can be used in an air atmosphere, hasonly 10 mmol/L bicarbonate and 40 mmol/L HEPES, butrequires 5 hours for sperm to capacitate with heparinowing to reduced levels of bicarbonate [21]. The mediumSp-TALP-H also is capable of supporting fertilization ofoocytes in an air atmosphere if needed (Parrish, personalobservation). The fertilization medium used is Fert-TALPand requires incubation in a 5% CO2 in air atmosphere [1].

At the time when HIS and then later heparin were beinginvestigated for treatment of sperm to increase IVF results,others were utilizing long incubation periods [22,23], oraddition of caffeine to BO medium and/or addition of cal-cium ionophore [24]. It was difficult at the time to under-stand how these different methodologies were enhancingthe ability of sperm to fertilize oocytes.

From the experiments using heparinwith bovine sperm,it is possible to come to a general understanding of theintracellular events of capacitation in the bovine. The firstobservation of importance is that heparin induces capaci-tation of bovine sperm rather than the acrosome reaction.This is best demonstrated by the requirement of at least a4-h incubation with heparin before sperm can either un-dergo a stimulus induced acrosome reaction from lyso-phosphatidlycholine [20], soluble zona pellucida proteins

[10,25], or penetrate zona intact bovine oocytes [20].Heparin must first bind to bull sperm before its ability toinduce capacitation [26,27] and the ability to capacitateresides in the charge dependent nature of this binding[26,28,29], because it can be inhibited by protamine sulfate[27]. The binding of heparin is to a series of bovine seminalplasma proteins (BSPs), that bind to epididymal sperm atejaculation [30]. These proteins include BSP-A1, BSP-A2,BSP-A3, and BSP-30-kDa. The BSPs interact with bothcholesterol and phospholipids in the sperm plasma mem-brane. After heparin binding, there is a loss of lectin bindingto bovine sperm, indicating the loss of sperm surfacecomponents [31,32]. The changes in surface componentslikely relate to a heparin-induced loss of the BSPs over timethat lead to a loss of membrane cholesterol and phospho-lipid [30]. Discussions of the role of cholesterol loss andmembrane modifications are discussed in Bailey [33],Leahy and Gadella [34], and Gadella [35].

As the loss of BSPs occur due to heparin binding tosperm, changes to sperm intracellular pH (pHi), intracel-lular calcium (Cai), and cAMP levels also happen because ofheparin. Investigations into the role of the intracellularchanges during capacitation of bovine sperm induced byheparin have been greatly helped by the effects of glucose.An interaction of heparin and glucose on sperm capacita-tion were first noted in Parrish et al. [18]. The effect ofglucose is not on heparin binding to sperm, because hep-arin binding was not affected by the presence of glucose[27]. Glycolysis of glucose or other similar substrates leadsto an acidification of bovine sperm that blocks heparin-induced capacitation [21]. The major effect of glycolysis isthat proton (Hþ) production acidifies the pHi of sperm,which opposes the heparin-induced alkalinization of pHi[21,36]. The effect of glucose can be circumvented byaddition of compounds that increase intracellular cAMP

J.J. Parrish / Theriogenology 81 (2014) 67–7370

such a 8-bromo-cAMP, isobutylmethylxanthine, andcaffeine or allowing sufficient time for sperm tometabolizeall the glucose present [18,27,37]. Ability of sperm tometabolize a substrate under closed incubation conditionshas rarely been taken into account during capacitation orfertilization experiments. A rise in sperm cAMP is neededfor heparin-induced capacitation [27], but blocking theeffects of cAMP with a specific inhibitor such as RP-cAMPdoes not prevent the increase in pHi [37], suggesting thecAMP increase is downstream of the change in pHi. Heparinthus binds to sperm, BSPs are lost along with membranecholesterol, pHi increases, and then cAMP increases.

Calcium is important to capacitation of sperm. Ejacu-lated bovine sperm have a very active plasma membranecalcium ATPase that extrudes calcium and maintains Cai inthe nanomolar range. Capacitation of bovine sperm withheparin requires extracellular calcium that is taken up bysperm and leads to a rise in Cai in the sperm head [38,39].Glucose blocks the uptake in calcium, but this can beovercome by the addition of cAMP modulators that in-crease cellular cAMP [38]. Interestingly, as heparin inducescalcium uptake by sperm, initially Cai in the head is low at102�13 nmol/L, but then increases to 184� 21 nmol/L by 4h of incubation, when sperm are capacitated [40]. Evidencesuggests that calcium uptake is critical for capacitationduring the first 2 hours of heparin exposure [39]. Duringthis time, the acrosome accumulates calcium and thusprevents a Cai increase in the cytoplasm. As the acrosomestore fills, Cai then increases; if a sperm does not come incontact with the appropriate stimulus, a spontaneousacrosome reaction and sperm death occur. The appropriatephysiologic stimulus would be the zona pellucida [10,25].During the acrosome reaction, the internal store would bereleased and a store-operated plasma membrane calciumchannel seems to be activated to increase calcium in thesperm cytoplasm [39]. The final increase in Cai is importantand is related to the physiologic ability to acrosome react.This was demonstrated in an experiment in which bovinesperm were loaded with Fura2 to measure Cai and thenincubated with and without heparin [41]. At 5 hours ofincubation, sperm were imaged for Cai in the sperm headand then exposed to soluble zona pellucida proteins andmonitored for an additional 15 minutes to determinewhether they acrosome reacted. Control, uncapacitatedsperm that did not or did acrosome react had 61 � 3 (n ¼207) and 78 � 7 (n ¼ 7) nmol/L Cai in the sperm head andwere not different (P > 0.05). In comparison, sperm incu-bated with heparin and did not or did acrosome react had102 � 9 (n ¼ 97) and 311 � 42 (n ¼ 58) nmol/L Cai in thesperm head (P < 0.05). Of the heparin-treated sperm thatacrosome reacted, the higher the Cai, the quicker spermacrosome reacted in response to zona proteins. Examina-tion of the anterior and posterior head Cai found evidencethat only spermwith Cai increases in the acrosome actuallyunderwent a zona pellucida–induced acrosome reaction.

Two other observations on heparin-induced capacita-tion are important [29]. The first is that a minimum of 10mmol/L bicarbonate is required in capacitation or fertil-ization medium. Sperm contain a soluble adenylate cyclase,present in the cytoplasm that is stimulated by bicarbonate.The increase in sperm cAMP during capacitation likely does

not occur in the absence of bicarbonate. The secondobservation is that BSA is almost always present in capac-itation and fertilization media. Heparin-induced capacita-tion does not require BSA, but BSA is required forcapacitated bovine sperm to undergo an acrosome reaction,either in response to lysophosphatidylcholine or zonaintact oocytes. Heparin likely leads to cholesterol loss fromsperm via the interaction with BSPs and so a potentialcholesterol acceptor like BSA is not needed to removecholesterol from sperm and modulate the dynamics of theplasma membrane. The exact role of BSA during the acro-some reaction remains unclear.

The question is often asked if heparin capacitates bovinesperm in vivo? Bovine oviduct fluid can capacitate bovinesperm in vitro in a time course similar to heparin and anactive component is a GAG similar to heparin, likely hep-aran sulfate [27,42]. The GAGs in the oviduct likely resideon oviduct epithelial cells as proteoglycans and interactwith sperm upon their binding to these cells in vivo.Interestingly, bovine oviduct fluid has low levels of glucose[42]. However, oviduct fluid does not increase sperm cAMPand so differences with heparin exist that may be explainedby multiple capacitation pathways activated by heparinin vitro [27]. The discovery of other protein-capacitatingagents in bovine oviduct fluid [43] suggests that GAGsmight not be the only agents responsible for capacitation.Heparin still capacitates bovine sperm in vitro and is amajor asset to IVF in the bovine.

After the changes in sperm pHi, Cai and cAMP, there is anactivation of protein tyrosine kinases and potentially inhi-bition of protein tyrosine phosphatases [33]. The changesmodulate sperm to be able to undergo an acrosome reac-tion when encountering the zona pellucida of the oocyte. Amodel that encompasses everything noted in the effects ofheparin on bovine sperm is reflected in the model ofcapacitation in Figure 2.

If we use the knowledge gained by examining howheparin capacitates bovine sperm, the other methods forcapacitating bovine sperm that were developed in the late1970s through the 1980s can be explained. The methodsthat used the HIS medium likely were displacing BSPs fromsperm, thus decreasing membrane cholesterol and alteringmembrane biophysical properties. The BO medium con-tained glucose and so was working in opposition to the HISeffects and likely delayed or prevented capacitation inmany males. Long incubation periods likely also involvedthe gradual loss of BSPs and associated membrane choles-terol. Male variation in affinity of the BSPs would likelyhave been important in finding a male whose spermwouldcapacitate before motility and viability of sperm decreased.The BO medium with addition of caffeine and/or a calciumionophore mimics the need for an increase in cAMP and Caiduring bovine sperm capacitation [24]. However, the use ofBO medium and cAMP modulation works better whenheparin is also included in the procedure [44,45]. The largenumber of publications related to bovine IVF since 2000make it impossible to exhaustively examine procedures.However, it is possible to make a general conclusion. Thereare two procedures that stand out and both use heparin.The first is the BO medium with cAMP, and heparin treat-ment. The second is modifications of the 1986 heparin

cAMP

H+

sAC

HCO3

-

pHi

HCO3

-

H+

Cholesterol

AcceptorBSP

Heparin

Binding

PKA

PTK Ptyr-Ptase

Protein Tyrosine Phosphorylation

(-)

(+)

(+)

Membrane

changes

(-)

(+)

(+)

(+)

Ca2+

Ca2+

Ca2+

Acrosome

Ca2+

ATP ADP

ATP ADP

(+)

(+)

Fig. 2. Proposed model of intracellular events during capacitation of bovine sperm by heparin. Ejaculated sperm bind seminal plasma proteins (BSP) at ejac-ulation. Heparin used during in vitro fertilization (IVF) binds to BSP and leads to their loss from the plasma membrane along with associated cholesterol andphospholipids. Other cholesterol acceptors in the capacitation/fertilization medium such as BSA are present and may also absorb membrane cholesterol. Thisleads to changes in the plasma membrane [35]. Intracellular events are then related to a heparin-induced decreased ability of sperm to extrude Ca2þ via acalcium-ATPase. A net uptake of calcium occurs and at first Ca2þ is taken up by the acrosome and intracellular Ca2þ (Cai) does not change until this store is filled.Once the acrosome is filled with Ca2þ, Cai begins to increase. Heparin binding also induces a net efflux of Hþ and presumed influx of HCO3

�. The model does notassume the changes in HCO3

� and Hþ are linked through for example a dual transporter. The net changes in HCO3� and Hþ increase intracellular pH (pHi). Sperm-

soluble adenylate cyclase (sAC) is stimulated by both HCO3� and increasing pHi. The resulting cAMP activates protein kinase A (PKA) and through cross-talk,

protein tyrosine kinases (PTK) are stimulated and protein tyrosine phosphatases (Ptyr–Ptase) are inhibited. A net increase in protein tyrosine phosphoryla-tion thus occurs. The rising Cai further stimulates sAC and the inhibition of Ptyr–Ptases. The capacitated sperm is thus primed to undergo a zona pellucida–induced acrosome reaction.

J.J. Parrish / Theriogenology 81 (2014) 67–73 71

paper [1], where heparin is simply added to the fertilizationmedium in different amounts along with sperm washingvia a Percoll or similar gradient [2,46].

4. Use of cryopreserved semen for IVF andadjustments for bull effects

Frozen-thawed semen was used in Parrish et al. [1] andwas critical to repeatable production of in vitro–produced(IVP) embryos. The sperm treatment procedure wasadapted from that used on ejaculated sperm [18] in whichsperm were pre-incubated with heparin. Thus sperm wereincubated for 15 minutes with 10 mg/mL heparin and thendiluted into the fertilization medium containing in vitro–matured oocytes. The short incubation time is owing tofrozen-thawed semen having reduced ability to surviveincubation when compared with unfrozen semen. Thisallowed a carryover of 0.2 mg/mL heparin during fertiliza-tion. It was the carryover level of heparin that was criticaland we later showed that fertilization rates with frozen-thawed sperm were heparin dose dependent [47]. After

this discovery, heparin was added directly to the fertiliza-tion medium. Using this approach, most oocytes arepenetrated by sperm within 4 to 6 hours with pronucleiforming between 6 and 10 hours after sperm addition[7,48]. It is possible then to adjust the percentage of oocytesfertilized by adjusting heparin levels. Generally these haveranged from 0.2 to 5 mg/mL, but higher levels can be used.Changing the amount of sperm added to the bovine IVFsystem also impacts the fertilization [47,49]. From years ofexperience, the rate of fertilization of in vitro mature oo-cytes is associated with rates of polyspermy [49]. Whenfertilization rates exceed 80%, polyspermy begins to in-crease, thus impacting eventual development rates. Theheparin system with frozen-thawed semen gives two crit-ical points at which to adjust fertilization rate for aparticular bull. You can change heparin levels and/or youcan change sperm concentrations to get optimal fertiliza-tion results that maximize sperm penetration of oocytes,but minimizes polyspermy. Because many straws can befrozen from a single bull ejaculate, it is then possible tofertilize oocytes with the same preparation of sperm and

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eliminate the male-to-male or ejaculate-to-ejaculatevariability.

5. Sperm washing procedures

Sperm preparation for IVF generally involves someprocedure to separate spermatozoa from seminal plasma,extender, and/or cryoprotectants. As pointed out, thenumber of sperm added to oocytes during IVF impacts thepercentage of oocytes penetrated by sperm and evenpenetration by multiple sperm in polyspermy. Semencontains both motile or viable sperm as well as nonmotilesperm that are usually dead and will not be capable offertilizing oocytes. To get repeatable results with IVF, it isessential to add the same number of sperm that have thepotential to participate in fertilization. With unfrozensemen, most sperm are motile/viable so simply deter-mining the concentration of sperm and adding a setamount is sufficient. Although frozen-thawed sperm pro-vide the advantage of using semen from the same bull andeven ejaculate, many sperm die in the cryopreservationprocess resulting in post-thawmotilities of 30% to 70% [46].Swim-up procedures were adapted to circumvent thisproblem [1]. In swim-up, sperm are layered at the bottomof a column of medium. The dense nature of semen inextender and cryoprotectant initially keeps these sperm atthe bottom. Over time sperm, begin to swim up out of theextender and cryoprotectant into the covering medium.Isolating just the covering medium provides a populationof sperm that is close to 100% motile or viable. This swim-up isolated population of sperm can simply be counted toadd a specific number of sperm to the IVF system. Althoughsimple in principle, the swim-up technique was difficult formany to implement. For example, if you get extenderassociated with the isolated sperm, the extender inhibitscapacitation and so fertilization. Further semen frozen forIVF use was often at 75 to 100 � 106/mL and much morethan the 15 to 30 � 106/mL used for commercial artificialinsemination. The number of sperm recovered from swim-up with commercial semen was then often quite low. APercoll gradient system similar to that used with humansperm was optimized for bovine semen between 1989 and1990 and shared with many laboratories for use in pre-paring bovine sperm for IVF. A comparison of the Percollapproach for sperm isolation with the swim-up methodwas described in Parrish et al. [46]. Recovery of motilesperm from frozen thawed semen was 9% � 1% for theswim-up approach but 40% � 4% for Percoll. The higherrecovery is why Percoll or something similar has beengenerally adopted. It was noted that at the same spermnumbers, IVF rates were higher for the swim-up isolatedsperm. Increasing the sperm concentration during fertil-ization, however easily, compensated for this deficit inPercoll-separated sperm.

6. Current status of IVF and embryo production

The IVF work done in the bovine has been valuable toboth the scientific and commercial interests. The work ofParrish et al. [1] now has more than 800 citations based asearch of the Web of Knowledge/Web of Science. The

bovine is one of the best models to study in vitro embryodevelopment. A search using PubMed found more than1000 publications related to bovine embryos from 2011 tothe present. The large supply of bovine oocytes obtainedfrom either slaughterhouse material or ovum pick-upprocedures with ultrasound-guided follicular aspirationmakes it possible to produce embryos in any quantitydesired. This is best illustrated by the worldwide statisticson IVP embryo production and transfer in 2011 [50]. Therewere 453,471 IVP embryos produced and 343,927 trans-ferred, the majority of which are in Brazil. Although it is notpossible to track down procedures used for IVF in all thelaboratories involved, most seem to be using some sort ofprocedure that involves heparin. The number of IVP-transferred embryos has been steadily increasing since2001 and is fast approaching the in vivo–produced andtransferred embryos of 572,342.

7. Conclusion

The development of the IVF system in the bovine did notoccur in a vacuum. The majority of the work was done inthe laboratory of N.L. First, but interaction with the labo-ratory of R.L. Ax also occurred. The First laboratory hadbeen assembled in the 1980s to develop cloning and genemodificationmethodology in the bovine, but individuals allwereworking in solving problems in specific areas. Many ofthe names will be recognized by researchers of today andinclude (listed in alphabetical order): F.L. Barnes, E.S.Critser, W.H. Eyestone, H.M. Florman, R.R. Handrow, M.L.Leibfried-Rutledge, D.L. Northey, J.J. Parrish, R.S. Prather,J.M. Robl, C.F. Rosenkrans, M.L. Sims, M.A. Sirard, J.L. Susko-Parrish, C.B. Ware, and M.A. Winer. The unique nature ofthe individuals and stimulating intellectual environmentmade the developments for the bovine IVF system possiblealong with many other contributions to science.

Acknowledgments

This work was supported by W.R. Grace & Co., NICHD,USDA-NRI, and the College of Agriculture and Life Sciences,University of Wisconsin-Madison.

References

[1] Parrish JJ, Susko-Parrish JL, Leibfried-Rutledge ML, Critser ES,Eyestone WH, First NL. Bovine in vitro fertilization with frozen-thawed semen. Theriogenology 1986;25:591–600.

[2] First NL, Parrish JJ. In vitro fertilization of ruminants. J Reprod FertSuppl 1987;34:151–65.

[3] Parrish JJ. Application of in vitro fertilization to domestic animals.In: Wassarman PM, editor. The Biology and Chemistry of Mamma-lian Fertilization, Volume II. New York: CRC Press; 1991. p. 111–32.

[4] Ball GD, Leibfried ML, Lenz RW, Ax RL, Bavister BD, First NL. Factorsaffecting successful in vitro fertilization of bovine follicular oocytes.Biol Reprod 1983;28:717–25.

[5] Critser ES, Leibfried-Rutledge ML, Eyestone WH, Northey DL,First NL. Acquisition of developmental competence during matura-tion in vitro. Theriogenology 1986;25:150.

[6] Sirard MA, Parrish JJ, Ware CB, Leibfried-Rutledge ML, First NL. Theculture of bovine oocytes to obtain developmentally competentembryos. Biol Reprod 1988;39:546–52.

[7] Leibfried-Rutledge ML, Critser ES, Parrish JJ, First NL. In vitromaturation and fertilization of bovine oocytes. Theriogenology1989;31:61–74.

J.J. Parrish / Theriogenology 81 (2014) 67–73 73

[8] Yanagimachi R, Usui N. Calcium dependence of the acrosome re-action and activation of guinea pig spermatozoa. Exp Cell Res 1974;89:161–74.

[9] Yanagimachi R. Mechanism of fertilization in mammals. In:Mastroianni L, Biggers J, editors. Fertilization and embryonicdevelopment in vitro. New York: Plenum; 1981. p. 81–182.

[10] Florman HM, First NL. The regulation of acrosomal exocytosis. I.Sperm capacitation is required for the induction of acrosome re-actions by the bovine zona pellucida in vitro. Dev Biol 1988;128:453–63.

[11] Parrish JJ. Capacitation of spermatozoa in vivo: physiochemical andmolecular mechanisms. J Mol Androl 1989;1:87–111.

[12] Brackett BG, Oh YK, Evans JF, Donawick WJ. In vitro fertilization ofcow ova. Theriogenology 1978;9:89.

[13] Brackett BG, Oh YK, Evans JF, Donawick WJ. Fertilization and earlydevelopment of cow ova. Biol Reprod 1980;23:189–205.

[14] Brackett BG, Bousquet D, Boice ML, Donawick WJ, Evans JF,Dressel MA. Normal development following in vitro fertilization inthe cow. Biol Reprod 1982;27:147–58.

[15] Brackett BD, Oliphant G. Capacitation of rabbit spermatozoa in vitro.Biol Reprod 1975;12:260–74.

[16] Lenz RW, Ball GD, Lohse JK, First NL, Ax RL. Chondroitin sulfate fa-cilitates an acrosome reaction in bovine spermatozoa as evidencedby light microscopy, electron microscopy and in vitro fertilization.Biol Reprod 1983;28:683–90.

[17] Lee CN, Ax RL. Concentrations and composition of glycosaminoglycansin the female bovine reproductive tract. J Dairy Sci 1984;67:2006–9.

[18] Parrish JJ, Susko-Parrish JL, First NL. Effect of heparin and chon-droitin sulfate on the acrosome reaction and fertility of bovinesperm in vitro. Theriogenology 1985;24:537–49.

[19] Bavister BD, Yanagimachi R. The effects of sperm extracts and en-ergy sources on the motility and acrosome reaction of hamsterspermatozoa in vitro. Biol Reprod 1977;16:228–37.

[20] Parrish JJ, Susko-Parrish JL, Winer MA, First NL. Capacitation ofbovine sperm by heparin. Biol Reprod 1988;38:1171–80.

[21] Parrish JJ, Susko-Parrish JL, First NL. Capacitation of bovine sperm byheparin: inhibitory effect of glucose and role of intracellular pH. BiolReprod 1989;41:683–99.

[22] Iritani A, Kasai M, Niwa K, Song HB. Fertilization in vitro of cattlefollicular oocytes with ejaculated spermatozoa capacitated in achemically defined medium. J Reprod Fertil 1984;70:487–92.

[23] Wheeler MB, Seidel Jr GE. Zona pellucida penetration assay forcapacitation of bovine sperm. Gamete Res 1987;18:237–50.

[24] Aoyagi Y, Fujii K, Iwazumi Y, Furudate M, Fukui Y, Ono H. Effects oftwo treatments on semen from different bulls on in vitro fertiliza-tion results of bovine oocytes. Theriogenology 1988;30:973–85.

[25] Florman HM, First NL. Regulation of acrosomal exocytosis. II. Thezona pellucida-induced acrosome reaction of bovine spermatozoa iscontrolled by extrinsic positive regulatory elements. Dev Biol 1988;128:464–73.

[26] Parrish JJ, Susko-Parrish JL, First NL. Capacitation of bovine sperm byheparin is correlated with 3H-heparin binding and is blocked byprotamine sulfate. Biol Reprod 1988;38(Suppl. 1):59.

[27] Parrish JJ, Susko-Parrish JL, Uguz C, First NL. Differences in the roleof cAMP during capacitation of bovine sperm by heparin or oviductfluid. Biol Reprod 1994;51:1099–108.

[28] Handrow RR, Boehm SK, Lenz RW, Robinson JA, Ax RL. Specificbinding of the glycosaminoglycan 3H-heparin to bull, monkey, andrabbit spermatozoa in vitro. J Androl 1984;5:51–63.

[29] Parrish JJ, Susko-Parrish JL, Handrow RH, Ax RL, First NL. Effects ofsulfated glycoconjugates on capacitation and the acrosome reactionof bovine and hamster spermatozoa. Gamete Res 1989b;24:403–13.

[30] Manjunath P, Therien I. Role of seminal plasma phospholipidbinding proteins in sperm membrane lipid modification that occursduring capacitation. J Reprod Immunol 2002;53:109–19.

[31] Medeiros CMO, Parrish JJ. Changes in lectin binding to bovine spermduring heparin-induced capacitation. Molec Reprod Develop 1996;44:525–32.

[32] Mahmoud A, Parrish JJ. Effect of capacitation on lectin binding tobovine sperm: flow cytometric analysis. Molec Reprod Develop1996;43:554–60.

[33] Bailey JL. Factors regulating sperm capacitation. Systems Biology inReproductive Medicine 2010;56:334–48.

[34] Leahy T, Gadella BM. Sperm surface changes and physiologicalconsequences induced by sperm handling and storage. Reproduc-tion 2011;142:759–78.

[35] Gadella BM, Luna C. Cell biology and functional dynamics of themammalian sperm surface. Theriogenology 2014;81:74–84.

[36] Vredenburgh-Wilberg WL, Parrish JJ. Intracellular pH of bovinesperm increases during capacitation. Molec Reprod Dev 1995;40:490–502.

[37] Uguz C, Vredenburgh WL, Parrish JJ. Heparin-induced capacitationbut not intracellular alkalinization of bovine sperm is inhibited byRp-adenosine-3’5’-cyclic monophosphorothioate. Biol Reprod 1994;51:1031–9.

[38] Handrow RR, First NL, Parrish JJ. Calcium requirement and uptakeduring capacitation of bovine sperm by heparin. J Exp Zool 1989;252:174–82.

[39] Parrish JJ, Susko-Parrish JL, Graham JK. In vitro capacitation ofbovine spermatozoa: role of intracellular calcium. Theriogenology1999;51:461–72.

[40] Parrish JJ, Vredenburgh WL, Lavin CA. Increases in bovine spermintracellular calcium (Cai) and pH (pHi) during capacitation. BiolReprod 1993;48(Suppl. 1):106.

[41] Parrish JJ, Susko-Parrish JL. Calcium increases in the anterior head ofbovine sperm during capacitation. Biol Reprod 2001;64(Suppl. 1):112.

[42] Parrish JJ, Susko-Parrish JL, Handrow RR, Sims MM, First NL.Capacitation of bovine spermatozoa by oviduct fluid. Biol Reprod1989c;40:1020–5.

[43] McNutt TL, Killian GJ. Influence of bovine follicular and oviduct fluidon sperm capacitation in vitro. J Androl 1991;12:244–52.

[44] Niwa K, Ohgoda O. Synergistic effect of caffeine and heparin on in-vitro fertilization of cattle oocytes matured in culture. Ther-iogenology 1988;30:733–41.

[45] Park CK, Ohgoda O, Niwa K. Penetration of bovine follicular oocytesby frozen-thawed spermatozoa in the presence of caffeine andheparin. J Reprod Fertil 1989;86:577–82.

[46] Parrish JJ, Krogenaes A, Susko-Parrish JL. Effect of bovine spermseparation by swimup or percoll on success of in vitro fertilizationand embryo development. Theriogenology 1995;44:859–69.

[47] First NL, Parrish JJ. Sperm maturation and in vitro fertilization.Dublin, Ireland: The 11th International Congress on Reproductionand Artificial Insemination; 1988:160–168.

[48] Eid LN, Lorton SP, Parrish JJ. Paternal influence of S-phase in the firstcell cycle of the bovine embryo. Biol Reprod 1994;51:1232–7.

[49] Parrish JJ, Eid L. In vitro fertilization and its relationship to bullfertility. 15th Technical Conference on Artificial Insemination andReproduction. Columbia, MO: National Association of AnimalBreeders; 1994. p. 68–73.

[50] Stroud B. The year 2011 worldwide statistics of embryo transfer indomestic farm animals. Report of the IETS 2012 Statistics and DataRetrieval Committee, International Embryo Transfer Society. 2012:1–25.