Embryo production and quality of Holstein heifers and cows supplemented with beta-carotene and tocopherol.

Abstract

The hypothesis was that the intramuscular injection (i.m.) of beta-carotene associated to tocopherol improves cow (n=86) and heifer (n=91) embryo production and quality. Time of estrus was synchronized in animals with an ear implant with 3 mg of norgestomet associated with an i.m. injection of 6 mg of norgestomet and 10mg of estradiol valerate (CRESTAR, Intervert International B.V., Boxmeer, Holland) and superovulated by 8 i.m. FSH/LHp injections (400 IU-heifers and 500 IU-cows) in decreasing concentrations at 12h intervals. Animals were inseminated 12 and 24h after observed onset of estrus and embryos recovered 7 days later. Animals were randomly allocated to one of three treatments: (1) vegetable oil vehicle (control), (2) 800 mg of beta-carotene and 500 mg of tocopherol (T800) and (3) 1200 mg of beta-carotene and 750 mg of tocopherol (T1200). Supplemental injections were given at the day norgestomet implants were inserted and at first superovulatory injection. An index (Embryo Quality Index or EQI) was proposed to more precisely evaluate embryo quality (excellent*1 + good*2 + regular*3 + poor*4 + degenerate*5 + unfertilized ova*5)/total. There was an interaction between physiological stage (heifer or cow) and treatment on EQI (P=0.01) and on the proportion of viable embryos (P=0.03), where both variables were improved in T1200 cows, but not in heifers. The average EQI for heifers and cows in control, T800 and T1200 were 2.6+/-0.3 and 3.6+/-0.3; 2.5+/-0.3 and 3.6+/-0.3; 2.9+/-0.3 and 2.7+/-0.3, respectively. The average total number of viable embryos was greater (P=0.01) in supplemented cows (3.5+/-1.1; 5.4+/-1.4 and 7.5+/-1.2 in control, T800 and T1200, respectively), but less (P=0.01) in heifers (7.5+/-1.2; 5.6+/-1.2 and 4.0+/-1.1 in control, T800 and T1200, respectively). Supplementation injections of beta-carotene associated to tocopherol improved embryo quality in superovulated Holstein cows, in the present experimental conditions and may be advantageous in similar embryo production systems. However, at dosages applied in the present experiment, this treatment should not be recommended for nulliparous heifers.

Full text

Page 1

Please cite this article in press as: Sales, J.N.S. et al., Embryo production and quality of Hol-
stein heifers and cows supplemented with ?-carotene and tocopherol, Anim. Reprod. Sci. (2007),
doi:10.1016/j.anireprosci.2007.04.001
ARTICLE IN PRESS
+Model
ANIREP-3346;No.of Pages13
Animal Reproduction Science xxx (2007) xxx–xxx
Embryo production and quality of Holstein
heifers and cows supplemented with
?-carotene and tocopherol
J.N.S. Sales∗, L.M.K. Dias, A.T.M. Viveiros,
M.N. Pereira, J.C. Souza
Department of Animal Science, Universidade Federal de Lavras, Lavras-MG,
Postal Code: 37200-000, Post-Box: 37, Brazil
Received 10 December 2006; accepted 3 April 2007
Abstract
Thehypothesiswasthattheintramuscularinjection(i.m.)of?-caroteneassociatedtotocopherolimproves
cow (n=86) and heifer (n=91) embryo production and quality. Time of estrus was synchronized in animals
with an ear implant with 3mg of norgestomet associated with an i.m. injection of 6mg of norgestomet and
10mg of estradiol valerate (CRESTAR®, Intervert International B.V., Boxmeer, Holland) and superovulated
by 8 i.m. FSH/LHp injections (400 IU-heifers and 500 IU-cows) in decreasing concentrations at 12h inter-
vals. Animals were inseminated 12 and 24h after observed onset of estrus and embryos recovered 7 days
later. Animals were randomly allocated to one of three treatments: (1) vegetable oil vehicle (control), (2)
800mg of ?-carotene and 500mg of tocopherol (T800) and (3) 1200mg of ?-carotene and 750mg of toco-
pherol (T1200). Supplemental injections were given at the day norgestomet implants were inserted and at
firstsuperovulatoryinjection.Anindex(EmbryoQualityIndexorEQI)wasproposedtomorepreciselyeval-
uate embryo quality (excellent*1+good*2+regular*3+poor*4+degenerate*5+unfertilized ova*5)/total.
There was an interaction between physiological stage (heifer or cow) and treatment on EQI (P=0.01) and
on the proportion of viable embryos (P=0.03), where both variables were improved in T1200 cows, but not
in heifers. The average EQI for heifers and cows in control, T800 and T1200 were 2.6±0.3 and 3.6±0.3;
2.5±0.3 and 3.6±0.3; 2.9±0.3 and 2.7±0.3, respectively. The average total number of viable embryos
was greater (P=0.01) in supplemented cows (3.5±1.1; 5.4±1.4 and 7.5±1.2 in control, T800 and T1200,
respectively), but less (P=0.01) in heifers (7.5±1.2; 5.6±1.2 and 4.0±1.1 in control, T800 and T1200,
respectively). Supplementation injections of ?-carotene associated to tocopherol improved embryo quality
in superovulated Holstein cows, in the present experimental conditions and may be advantageous in similar
∗Corresponding author. Tel.: +55 35 3829 1686; fax: +55 35 3829 1231.
E-mail addresses: znlogan@yahoo.com.br, jcamisao@ufla.br (J.N.S. Sales).
0378-4320/$ – see front matter © 2007 Elsevier B.V. All rights reserved.
doi:10.1016/j.anireprosci.2007.04.001

Page 2

Please cite this article in press as: Sales, J.N.S. et al., Embryo production and quality of Hol-
stein heifers and cows supplemented with ?-carotene and tocopherol, Anim. Reprod. Sci. (2007),
doi:10.1016/j.anireprosci.2007.04.001
ARTICLE IN PRESS
+Model
ANIREP-3346; No.of Pages13
2
J.N.S. Sales et al. / Animal Reproduction Science xxx (2007) xxx–xxx
embryo production systems. However, at dosages applied in the present experiment, this treatment should
not be recommended for nuliparous heifers.
© 2007 Elsevier B.V. All rights reserved.
Keywords: Cattle; ?-carotene; Tocopherol; Embryo transfer; Superovulation
1. Introduction
The compound, ?-carotene, is a lipid soluble isoprenoid pigment naturally produced by plants
and photosynthetic organisms. This carotenoid is the natural vitamin A precursor in feed and is
converted to retinol (its storage form) in the liver and used by tissues as retinal (Bendich and
Olson, 1989). Tocopherol is an organic lipid soluble and biologically active alcohol responsible
for most of feed vitamin E activity (Bregelius and Traber, 1999; Metre and Callan, 2001). Both
vitamins are potent anti-oxidative agents, so that, any substance, present in lesser concentrations
compared to the oxidizable substrate delays or inhibits oxidation of this substrate in an efficient
manner (Sies and Stahl, 1995).
Beneficial effects of ?-carotene in the reproduction has been reported for various species. Par-
enteral administration of ?-carotene in mice (Chew and Archer, 1983) and pigs (Brief and Chew,
1985; Kostoglou et al., 2000) decreased embryonic mortality and increased litter size. In dairy
cows, supplemental dietary ?-carotene decreased retained placenta and endometritis incidence
compared to no supplementation or only vitamin A supplementation (Michal et al., 1994).
Thebestdescribedroleoftocopherolinmammalianspeciesreferstoitsantioxidativefunction,
by cellular control of lipid peroxidation explained by effects on inflammatory responses, immune
system, striated musculature and reproduction (Metre and Callan, 2001). Vitamin E treatment
accelerates uterine involution in cows with metritis (Harrison et al., 1984). Vitamin E protects the
early embryo from the effects of ambient heat stress (Arechiga et al., 1994), a cytotoxic event that
is probably mediated by free radicals (Loven, 1988).
Embryo transfer is a biotechnique that allows embryo collection from a donor female and
subsequent transfer to recipients to carry a pregnancy to term (Reichenbach et al., 2002). Embryo
qualityisveryimportantinembryotransferprograms,becauseitreflectsdirectlyuponpregnancy
outcome after embryo transfer (Abe et al., 2002). Various factors may affect embryo quality.
Nutrition is very important but it is still not well studied or understood. Among nutritional factors
that could affect embryo quality, antioxidants, such as, ?-carotene and tocopherol show great
potential. The injectable form used in the present study was used to ensure delivery and avoid the
usual potency loss that occurs in feed due to humidity, light, heat and management errors, thus,
targeted plasma concentrations would be reached and maintained (Herdt and Stowe, 1991).
In the present study ?-carotene was studied along with tocopherol with the objective of assess-
ingsynergisticeffectsofthetwosubstances,forthelatterisalsorecognizedasanexcellentnatural
antioxidative agent that protects carotenoids in the animal body (Weiss et al., 1994; Lal Thankur
and Srivastava, 1996). This association improves reproductive efficiency in many animal species.
However, any effect on embryo quality in superovulated cattle has yet to be assessed. These two
antioxidants were given to superovulated donor females in an attempt to decrease the oxidative
effects in animals that are likely to be undergoing hormonal stress and are also producing a large
number of corpora lutea, which, according to Haliloglu et al. (2002), have significant ?-carotene
concentrations which may be necessary for steroidogenesis. So, the rationale was that the extra
corpora lutea may concentrate ?-carotene and possibly tocopherol.

Page 3

Please cite this article in press as: Sales, J.N.S. et al., Embryo production and quality of Hol-
stein heifers and cows supplemented with ?-carotene and tocopherol, Anim. Reprod. Sci. (2007),
doi:10.1016/j.anireprosci.2007.04.001
ARTICLE IN PRESS
+Model
ANIREP-3346; No.of Pages13
J.N.S. Sales et al. / Animal Reproduction Science xxx (2007) xxx–xxx
3
The objective of the present study was to evaluate the effect of an intramuscular injection of ?-
carotene combined with tocopherol on embryo production and quality of superovulated Holstein
heifers and cows. Additionally, a novel embryo grading system is proposed, offering an option to
the current IETS embryo classification system.
2. Material and methods
2.1. Donors, animal facilities and diets
The present experiment was conducted from October 2003 through September 2004, at the
Fazenda Vista Alegre III, in the county of Curvelo in the central-north region of the state of
Minas Gerais, at 633 meters of altitude, south latitude 18◦45?56??and west Greenwich longitude
44◦27?27??.Treatmentsforthedifferentphysiologicalstage(heiferorcow)wereappliedsimultane-
ously at various time periods, and a balanced proportion of physiological stage and treatment was
maintainedthroughoutthetrial.Heifers(n=91)andcows(n=86)averaged(means±S.D.)21±2
and71±29monthsofage,363±27and687±128kg,3.5±0.3and3.4±0.4ofbodycondition
(on a 1–5 scale; Wildman et al., 1982), respectively. Lactating cows averaged (means±S.D.)
113.1±45.1daysinmilkand23.2±2.7kgdailymilkproductionacrossalltreatments(nostatis-
tical differences were found between treatments). Pastures were mainly Brachiaria brizantha and
Cynodon dactylon (tifton). Heifers were maintained in tifton paddocks and supplemented daily
with tifton hay and balanced concentrate (corn bran, soybean meal, dried citrus, lime, dicalcium
phosphate, sodium chloride and mineral mix). As for cows, heifer diet was designed to meet
100% of NRC requirements (NRC, 1989). Cows were kept in a free-stall system. Brachiaria
brizantha small paddocks served as rest areas and cows received elephant grass silage (Pennise-
tum purpureum), tifton hay and concentrate (same ingredients as the heifer concentrate added of
cottonseed).
2.2. Estrous synchronization, superovulation and artificial insemination
On Day 0 (day of implant administration), when donors were evaluated for reproductive
soundness by rectal palpation, subcutaneous ear implant inserted with 3mg of norgestomet
(CRESTAR®, Intervert International B.V., Boxmeer, Holland) was placed, plus an i.m. injec-
tion of 6mg of norgestomet, and 10mg of estradiol valerate. On Day 5, superovulation was
initiated. In the superovulation protocol 500 IU (cows) and 400 IU (heifers) of FSH (PLUSET®
I.F.Serono,Rome,Italy),weredistributedindecreasingdosagesof100,100,75,75,50,50,25,25
IU(cows)and80,80,60,60,40,40,20,20IU(heifers)during4days,at12hintervals.Luteolysis
was induced by a single i.m. injection of 500?g of DL cloprostenol (SINCROSIN®, Vallee S.A.,
Montes Claros, Brazil). The ear implants were removed on the seventh FSH injection (Day 8)
and estrus was observed on Day 9. Animals in estrus were inseminated by a single experienced
technician 12 and 24h after the onset of estrus. Animals were flushed on Day 16 (7 days after
estrus). Only uterine flushes yielding at least two ova/embryos were included in the statistical
analysis to ensure that animals were actually superovulated.
2.3. Treatments
Donors(n=177)wereallocatedtotreatmentsrandomlyastheyenteredthechutetobeevaluated
for reproduction soundness on Day 0. The first i.m. supplementation was given on Day 0 and the

Page 4

Please cite this article in press as: Sales, J.N.S. et al., Embryo production and quality of Hol-
stein heifers and cows supplemented with ?-carotene and tocopherol, Anim. Reprod. Sci. (2007),
doi:10.1016/j.anireprosci.2007.04.001
ARTICLE IN PRESS
+Model
ANIREP-3346; No.of Pages13
4
J.N.S. Sales et al. / Animal Reproduction Science xxx (2007) xxx–xxx
secondonthefirstdayofsuperovulationorDay5.Treatmentswereasfollows:(1)donorsreceived
a10mLi.m.injectionofvegetableoil(control;n=59,29cowsand30heifers),(2)donorsreceived
800mg of ?-carotene and 500mg of tocopherol (T800; n=59, 28 cows and 31 heifers) and (3)
donors received 1200mg of ?-carotene and 750mg of tocopherol (T1200; n=59, 29 cows and
30 heifers).
2.4. Embryo transfer
Embryos were collected from donors seven days after the first AI by a non-surgical procedure
(Wright, 1981) with 1000mL of Dulbecco’s modified phosphate buffered saline (DMPBS-
Nutricell®, Campinas, Brazil). Embryos were evaluated by consensus of two experienced
veterinariansaccordingtoLindnerandWright(1983).Theembryogradingsystemrecommended
by IETS (International Embryo Transfer Society, 1999) may be influenced by a variable degree
of subjectiveness. However, a very comprehensive study, done by Hasler (2001), demonstrated
clearly that the traditional grading system (Lindner and Wright, 1983) is efficient and is related
directly to embryo survival after transfer, more so than any other variable considered in their
study.
An index (Embryo Quality Index or EQI) was proposed to more precisely evaluate embryo
quality(excellentembryos *1+good*2+regular*3+poor*4+degenerate*5+unfertilized
ova*5)/total embryos recovered. For example: if a uterine flush yielded four excellent, three good
and one degenerate the index would be (4×1+3×2+0×3+0×4+1×5)/8=1.87. Values
closer to one indicate greater quality. The proportion of viable embryos (VIAVP), based on the
same classification, was defined as the sum of viable embryos (excellent+good+regular+poor),
divided by the total number of ova/embryos.
2.5. Blood sampling and serum β-carotene analysis
Blood samples were collected by coccygeal vessel venipuncture in 12 donors (four from
each treatment), for ?-carotene serum concentration determination. Samples were collected from
each animal on Days 0, 5 and 16. Samples were centrifuged (2000×g) for 10min and freezer
storedincryogenictubes(Cryogenicvial-Corning®)at−21◦C.Concentrationof?-carotenewas
determined by high pressure liquid chromatography described elsewhere (Njeru et al., 1992).
2.6. Statistical analysis
The experimental design was a 2 by 3 factorial, where two physiological stages (cows or
heifers) were compared across three treatments (control, T800 and T1200). The variables EQI,
VIAVP, total ova/embryos and viable embryos, were analyzed by the GENMOD procedure of
SAS (SAS Technical Report -243, 1993) and, as determined by the UNIVARIATE procedure of
SAS (SAS, 1995), characterized as a Poisson distribution. Because the Poisson curve implies in
over dispersion, the GENMOD procedure was applied with a scale parameter (pscale option) to
account for it and adjust the model adequately. Least square means (generated by the GENMOD)
were compared by orthogonal contrasts. Fertilization rate data were analyzed by chi-square with
the PROCFREG procedure of SAS. The following model was used:
Yij= μ + Pi+ Tj+ P × Tij+ eij

Page 5

Please cite this article in press as: Sales, J.N.S. et al., Embryo production and quality of Hol-
stein heifers and cows supplemented with ?-carotene and tocopherol, Anim. Reprod. Sci. (2007),
doi:10.1016/j.anireprosci.2007.04.001
ARTICLE IN PRESS
+Model
ANIREP-3346;No.of Pages13
J.N.S. Sales et al. / Animal Reproduction Science xxx (2007) xxx–xxx
5
where μ=constant associated to all observations; Pi=effect of physiological stage i (i=heifer
and cow); Tj=effect of treatment j (j=control, T800 and T1200); P×Tij=interaction between
physiologicalstageandtreatment;eij=experimentalerrorassociatedtoYij,which,byhypothesis,
has a Poisson distribution where the mean is equal the variance.
3. Results
Supplementary injections (800mg or 1200mg) on Days 0 and 5 increased ?-carotene con-
centrations from 3.4±1.1 and 5.1±1.1?g/mL before injection (Day 0) to 9.6±0.9 and
10.9±3.4?g/mL (Day 16), respectively (Fig. 1). Similar findings were observed by Arechiga
et al. (1998a,b), where ?-carotene concentrations after three injections remained elevated for 19
days after the first injection.
Of the 177 donors only 132 (65 cows and 67 heifers) were considered for analysis, based on
the production of at least two ova/embryos. Because of lack of response to the superovulation
protocol, 34 animals (5 heifers and 6 cows in control, 6 heifers and 8 cows in T800 and 4 heifers
and 5 cows in T1200) were removed from the trial. An additional group (n=11) were not used
because they were unsuitable for insemination, as they presented abnormal mucous discharge or
because of problems such as, implant loss or death. None of these events were due to treatment
effects as they were included in an initial broader model and withdrawn for the final analysis.
Treatments did not affect embryo quality index (Table 1). However, there was a physiological
stage*treatmentinteractioneffectonembryoquality(Table1).Embryoqualityimprovedincows
that received the greatest vitamin dose (T1200). In contrast, the T1200 dose decreased embryo
quality in heifers, compared to control. Physiological stage affected embryo quality (Table 1)
which was better in heifers than in cows. The embryo quality index scores may be compared
to the average numbers of embryo as graded in the traditional method (Table 2). Note that the
main effect of physiological stage, which affected the EQI, was not detectable with the traditional
grading system, except for unfertilized ova. The proportion of viable embryos (VIAVP) also was
not affected by treatment but there were effects of physiological stage and of the interaction
Fig. 1. Mean ?-carotene serum concentrations (determined by HPLC) in animals receiving i.m. injections of vegetable
oil (?), 800mg of ?-carotene (?) and 1200mg of ?-carotene (?) on Days 0 and 5 of the experimental. Arrows represent
supplementary injection days. The concentrations of ?-carotene were different (P<0.05) between all treatments on Day
5 (*) and between control and the other treatments on Day 16 (**). Values were averaged between physiological stages
(because values did not differ significantly, P>0.05 between physiological stages). Only samples from four heifers and
five cows were used in this analysis.