EFFECT OF NUTRITION ON ENDOCRINE PARAMETERS, OVARIAN

PHYSIOLOGY, AND OOCYTE AND EMBRYO DEVELOPMENT

M.P. Boland, lv3P. Lonergan’*’ and D. O’Callaghanz3

Departments of ‘Animal Science & Production, and 2Animal Husbandry & Production,
and 3Conway Institute of Biomolecular and Biomedical Research, University College
Dublin, Beltield, Dublin 4, Ireland

Received for publication: June 6, 2000

Accepted: September 5, 2000

ABSTRACT
Reproductive efficiency in high yielding dairy cows has decreased over the past
50 years, despite significant gains in genetic selection for increased milk output. One
possible reason for this decline has been a change in the nutritional intake to meet the
increased energy and protein demands for higher milk production. Excess energy
intake in sheep will lead to significant reductions in progesterone concentrations; the
effects in cattle are not so clear. Nutrition, unless radically changed, will have little
effect on gonadotropin concentrations in ruminants, and this is in contrast to the
situation for pigs and for primates, where very short-term nutritional changes manifest
themselves in altered gonadotropin secretion. Cattle with reduced energy intake have
smaller dominant follicles and more three-wave cycles, compared with animals on
higher feed intakes. One of the main areas where nutrition influences reproductive
efficiency is at the level of embryo production. Several studies indicate that excess
energy intake reduces the response to superovulation and also decrease the yield of
embryos and alters expression of some gene constructs within the developing embryo.
The mechanism of this effect is not clear but indications are that the quality of the
oocytes may be compromised. Indeed recent data indicate that nutritional changes
around the time of mating may have detrimental effects on the establishment of
pregnancy in heifers. Thus, nutritional balancing is critical for high-yielding dairy
cows, in particular. The challenge remains to modify nutritional and management
strategies in such cows to maintain the levels of production made possible by genetic
selection and still maintain an acceptable level of fertility.
0 2001 by Elsevier Science Inc.
Key words: reproductive efficiency, nutrition, oocyte, embryo

INTRODUCTION

Recent genetic improvement in dairy cows has led to a substantial increase in

milk yield, which has been associated with a decrease in reproductive performance (7).
Reduced fertility is particularly obvious in cows where milk yields are above 6000 kg
per lactation (52. 66) and in cows fed in excess during the previous dry period (42).
Early embryo mortality is’a significant cause of reproductive failure in ruminants and
part of this may be related to nutritional influences around the time of mating (22).
Although the final manifestation of a detrimental effect of nutrition on fertility may be
the death of the embryo, it is not clear if nutrition affects embryo quality through

Theriogenology 55: 1323-1340,200l 0093-691X/01/$-see front matter

Q 2001 Elsevier Science Inc. PII: SOO93-691X(01)00465-X
1324 Theriogenology

changing the follicular environment, the developmental capacity of the oocy-te itself or
through changes occurring during early embryo development.
Nutrition can influence reproductive function in ruminants. However, the
relationship between nutrition and reproduction is complex and responses are often
quite variable and inconsistent. In the case of lactating dairy cows, inadequate
nutrition in the short term, or as a consequence of a prolonged depletion of body
reserves during early lactation, can have significant deleterious effects on resumption
of ovarian activity postpartum, conception rate and infertility. Deleterious effects of
excessive nutrition around the time of mating on embryo development, are becoming
evident both in unsuperovulated (21) and in superovulated cattle (57, 68, 96). The
objective of this paper is to review the effects of nutrition on some endocrine
parameters, follicular development, oocyte quality and embryonic development with
particular emphasis on effects in ruminants.

ROLE OF NUTRIENTS IN FARM ANIMALS

In ruminants, the digestive process is a complex process that resembles a

system within a system, with most of the diet consumed acting as a nutrient supply for
ruminal microflora. These in turn produce energy and protein components that can be
digested and absorbed. The rumen is populated by many types of micro-organisms.
Some digest complex carbohydrates, including cellulose-based carbohydrates, and
produce volatile fatty acids such as acetate, propionate and butyrate. Propionate is the
main energy substrate used by the ruminant and is converted to glucose in the liver.
The relative amounts of each volatile fatty acid produced is diet dependent, with
roughage-type feeds encouraging the production of acetate and cereal-based diets
encouraging the production of propionate. Thus, diet type can alter the nutrients
available for productive purposes.

Animals require protein as a source of essential amino acids and (in the case of
ruminants) as a nitrogen source for rumen microflora. The quality of the protein in a
feed is dependent on its amino acid profile and digestibility. Dietary protein is
categorized as rumen degradable or undegradable on the basis of the ability of the
microbes to hydrolyze the protein in the rumen. The protein requirement of an animal
is dependent on its physiological status and level of production. Essential amino acids
must be supplied in the diet of monogastrics, but rumen microbes are themselves the
main source of amino acids for ruminants. Ruminants are also capable of reducing
protein loss by recycling urea, a product of protein metabolism that is normally
excreted. Thus, some urea can be recycled to the rumen when the diet is low in
nitrogen. Surplus amino acids are deaminated and the nitrogen is excreted via the liver
and kidneys, mainly as urea in the urine. Excess ammonia is conjugated to urea and
then excreted. Thus, high urea levels are consistent with excess protein intake,
possibly with concomitant energy shortage, and are likely to be associated with high
levels of ammonia in circulation.

EFFECT OF DIETARY UREA ON FERTILITY

High dietary protein, resulting in high concentrations of urea nitrogen in plasma

and milk (>190 mg/L) has been associated with decreased fertility in dairy cattle (12,
24, 36). One suggestion is that ths is due to an altered uterine environment (24). In
Theriogenology

sheep, Fahey et al. (26) reported that despite high dietary urea and blood urea
concentrations, there was no effect of dietary urea on ovulation rate in donor or
recipient ewes. However, embryo quality in donors was reduced as fewer embryos
with more than eight cells were recovered at Day 4 from urea-treated ewes. The diet
offered to recipients had no effect on embryo survival. Thus, it was suggested that the
effects of urea on embryo quality are likely to be due to alterations in the oviduct
environment or deleterious changes in the follicle, rather than changes in the uterine
environment. This has been supported by recent data in cattle (33) showing no
difference in pregnancy rates at Day 35 after transfer of good quality in-vitro produced
embryos to recipients on different levels of dietary urea.

Another manifestation of excess urea in the circulation is the birth of abnormally

large offspring (100). Ewes fed excess amounts of urea from 2 1 days before mating to
Day 63 of gestation resulted in oversized lambs at birth (62). It was suggested that this
effect occurred through embryonic exposure to high concentrations of ammonia in the
reproductive tract. As well as these effects observed in-vivo, links have also been
established between high levels of ammonia and poor embryo development m-vitro in
some culture systems (3 1).

The results of these and other studies (69,70) indicate that the measurement of
plasma urea concentration around the time of insemination or embryo transfer is of
little value for predicting subsequent fertility. Adequate energy intake in association
with dietary crude protein intake may be critical and is an area that requires further
investigation. These studies suggest that the deleterious effects of increased plasma
urea identified are likely to occur at the level of the oocyte within the follicle. We
propose that this results in the development of poor quality embryos, rather than the
effects of urea being primarily due to disruptions in the uterine environment. This
highlights the importance of nutrition in the pre-ovulation period (possibly for many
weeks) on fertility outcome.

NUTRITION AND GONADOTROPIN SECRETION

Energy status is generally considered to be the major nutritional factor that

influences reproductive processes, with prolonged low energy intake impairing fertility.
In sheep, poor nutrition, which results in lower ovulation rates, is associated with
decreased LH pulse frequency, which is likely due to inadequate hypothalamic G&I-I
secretion (77). In cattle, a strong correlation between negative energy balance in early
lactation and resumption of ovulation postpartum is evident (13). While ovulation may
not occur in animals on low dietary intakes, follicle growth and atresia will occur.
Such follicle wave turnover without ovulation is often evident in postpartum beef cows
in poor body condition (87). The practical significance of this occurrence is a
lengthening of the calving to first ovulation interval, and often an extension in the
calving to conception interval. Long-term restriction in feed intake has been shown to
induce anestrus in cattle (80), due to insufficient circulating LH (79). These effects,
however, are not immediately evident and dietary restriction of several months may be
required to prevent follicle growth and ovuIation.
1326 Theriogenology

In contrast to the situation in monogastrics, effects of short-term dietary

restriction on LH pulse patterns are more difficult to observe in ruminants. In ewes,
restricted dietary intake resulted in no change in LH secretion (3) or a relatively small
reduction in LH pulse frequency (77,78) when diets were restricted for approximately
3 weeks. The FSH is essential for follicular growth and ovulation (27). Yet there is
little evidence of an effect of nutrition on plasma FSH concentrations. However,
Mackey et al. (53) have shown that short-term restriction of dietary intake to
approximately 40% of maintenance energy requirements increased FSH in heifers
compared with those offered diets of twice maintenance energy requirements. This
trend was repeated in ovariectomized heifers on similar diets, suggesting that the
effects are mediated at least in part by changes at the level of the pituitary and not
solely due to alteration in steroid feedback effects. Thus, nutritional effects on
gonadotropin secretion in cattle are relatively minor unless dietary restrictions persist
for extended periods of time.

NUTRITION AND PROGESTERONE CONCENTRATIONS

Feed intake in sheep can influence the concentration of progesterone, with a

strong negative correlation between dietary intake and progesterone concentrations
(61, 78). This effect of intake on circulating progesterone concentration may be due
to an increase in the rate of catabolism of progesterone and in hepatic circulation at
higher feeding levels (74). Progesterone, through its negative feedback effects, can
affect LH pulse frequency and is also thought to play an important role in oocyte
maturation and in early embryo development (4 1, 6 1).

Feeding sheep on an ad-libitum basis consistently reduces progesterone

concentrations compared to restricted feeding (71), but the results in cattle indicate
that this effect is more variable. Ad-libitum feeding in heifers increased (60)
decreased (92) or had no effect (86) on progesterone concentration when compared
with restricted feeding. Low progesterone post breeding can reduce fertility (44).
However, as steroids are selectively stored in fat, any dietary regimen that results in
fat mobilization will result in the release of stored progesterone. This may account for
some of the increased progesterone evident in animals on low dietary intakes.
Recently, concentrations of progesterone and embryonic interferon-tau have been
positively correlated (56). Thus, minor changes in maternal progesterone
concentrations during the initial period of embryo development may alter the secretion
of this anti-luteolytic agent and may be critical to embryo survival.

In sheep, overfeeding that reduced circulating progesterone concentrations also

reduced pregnancy rates (73) and decreased both the rate of development and viability
of embryos (16). In cattle, Mann et al. (56) reported that the timing of the
progesterone rise after ovulation is of importance to the development of the embryo.
These authors showed that a delayed rise in progesterone was associated with smaller
and potentially less viable embryos at Day 16. A more recent experiment in beef
heifers showing the detrimental effect of an acute reduction of energy intake
immediately atter insemination on embryo survival, failed to find an association
between progesterone concentration early in the estrous cycle and embryo survival
(22). Peripheral concentrations of progesterone on Days 0 and 1 after the LH peak
are important for embryo survival in sheep (6). This presumably modifies follicular
Theriogenology 1327

maturation and oocyte quality. However, others have suggested that the effect of
progesterone on embryo development is acting primarily through the effect of
progesterone in the uterus (4, 48). Increased concentrations of progesterone during
the luteal phase before and after breeding have been associated with higher pregnancy
rates (12). All of these experimental results are based on the study of peripheral
concentrations of progesterone. However, experiments studying the effect of nutrition
on peripheral (jugular) and local (ovarian vein and endometrium) concentrations of
progesterone could not demonstrate any relationship between these measurements (2,
48). Thus, the use of jugular vein concentration of progesterone alone as an indicator
of the effect of nutrition on embryo development must be employed with caution, as
embryo survival may be more related to concentrations of progesterone in the ovarian
vein and the endometrium than to circulating concentrations.

NUTRITION AND OVARIAN FUNCTION

The ability of nutrition to alter the ovulation rate and lambing rate of ewes is well
known, where a rapid improvement in body condition is usually associated with an
increased ovulation rate and litter size (15). Alterations in ovulation rate may be
related to the cell entry rate of glucose in animals on a high plane of nutrition. Dietary
supplements containing high energy and protein have been shown to increase the
ovulation rate in ewes (19). Similarly, increases in ovulation rate were reported when
glucose was infUsed directly (20,93). Thus, it is likely that short-term energy supply is
directly involved in follicle recruitment (35) and perhaps also in follicle growth;
however, this effect may be of short duration when diet level is altered.

Dietary restriction has been shown to alter follicle growth characteristics in

cattle (65) and in superovulated sheep (98). Murphy et al. (65) reported that heifers
on a low dietary intake had reduced size and persistence of the dominant follicle
compared with animals offered higher energy intakes. Acute nutritional restriction (0.4
times maintenance) for about 12 days decreases the growth rate and maximum
diameter of the dominant follicle and induces failure of the dominant follicle to ovulate
after induced luteolysis with prostaglandin (54). Several studies have shown that
feeding fat altered the growth pattern of follicles and this effect is somewhat
independent of energy intake(S9). Supplemental fat increased the number of follicles
(11, 51) and increased the size of the preovulatory follicle (50). This increased follicle
size may have beneficial effects on both oocyte quality (47) and on corpus luteum
Iunction (59) resulting ultimately in higher pregnancy rates.

In the case of superovulated heifers, Nolan et al. (68) reported an increase in

follicle numbers a&r stimulation with exogenous FSH in heifers offered a low dietary
intake compared with heifers on a high dietary intake. This difference in response was
predominantly due to an increase in the number of follicles in the 7 to 10 mm size
range when measured around the time of the LH surge. However, this trend was not
repeated when ovulation rates were recorded a&r superovulation (68). In the case of
ewes superovulated with FSI-I, a lower ovulation rate was recorded in ewes offered
diets of half of the maintenance energy requirements, compared with ewes offered
diets of twice the maintenance energy requirement (98). In a later trial, the ovulation
rates of ewes superovulated on similar diets but using a different gonadotrophin
preparation were not different (99). Thus, it is clear that dietary intake can, under
1328 Theriogenology

certain conditions, alter the growth characteristics of follicles. However, the effect of
dietary intake on the’number of follicles growing in response to stimulation with a
fixed dose of FSH during superovulation is less consistent and thus, in that case it is
more difficult to draw firm conclusions.

LEPTIN AS A METABOLIC MARKER AFFECTING REPRODUCTION

Leptin is a peptide (secreted by white adipocytes) that plays a role in the

regulation of body weight and food intake. It has recently been implicated in the
interaction between nutrition and fertility (5, 14, 17, 34). Leptin receptors have been
identified in many areas of the brain and in many other tissues, including the ovary
(89). In the ewe, mRNA for leptin receptor is differentially expressed in well-fed and
feed-restricted animals (23). The current hypothesis is that leptin acts as an appetite
transducer and as a satiety factor. In rats, increasing leptin in circulation, decreased
rnRNA for neuropeptide-Y (which stimulates food intake), and increased mRNA for
corticotrophin-releasing hormone (an inhibitor of food intake; 84). Feed intake of
lambs is reduced in response to infused leptin. Thus, it is interesting to speculate that
dairy cows that are overfed in the dry period have reduced feed intake in early lactation
and that these effects on feed intake may be mediated by increased leptin
concentrations.

Administration of leptin has been shown to stimulate GnRH and LH secretion

in pituitary cells in-vitro, and to a lesser extent, FSH. In mice, leptin increases the
number of follicles on the ovary (8). A possible mechanism for the regulation of
reproduction by leptin would involve leptin binding to the B-endorphin neuron, which
impacts on the GnRH neuron. The B-endorphin neurons also impact on the
neuropeptide-Y neurons, which could be involved in satiety control. Leptin may also
have an effect locally, acting within the ovary to regulate follicle size and possibly
oocyte quality. Spicer et al. (85) suggested that leptin could act as a metabolic signal
to the reproductive system after observations that leptin can attenuate insulin-induced
steroidogenesis of bovine granulosa cells without affecting cell proliferation. In
addition, hypothalamic leptin receptor expression is greater in feed-restricted ewes
than in well-fed ewes (38). Thus, leptin may act as a general modulator of
reproduction by regulating appetite and feed intake and also by having direct effects on
the reproductive axis, possibly at several levels, but largely by central inhibition of
neuropeptide-Y.

NUTRITION AND OOCYTE QUALITY

While structural differences have been reported in oocytes from superovulated

compared with unstimulated heifers (7), few studies report effects of nutrition on
oocyte quality in detail. McEvoy et al. (61) reported that a higher proportion of ova
from ewes on a low diet were considered viable when compared with those produced
in ewes on a high diet, We determined the effect of dietary intake on oocyte
morphology in sheep oocytes using electron microscopy (69). Structural changes in
the degree of detachment of interchromatin-like granules within the oocyte nucleolus
were observed in oocytes collected from superovulated compared with control ewes.
However, no differences in oocyte morphology were evident in oocytes collected from
Theriogenology

ewes offered a low dietary intake (half maintenance energy requirement) compared
with a high diet (double maintenance energy requirement).

With cattle oocytes, restricting energy intake before slaughter enhanced the
subsequent in-vitro development of the oocytes from small follicles (62). Yaakub et
al. (98) stimulated heifers with FSH and fed them a low (silage alone) or high (silage
plus 6 kg concentrates) diet before slaughter. The cleavage rate was improved
although no significant difference was evident in the in-vitro blastocyst formation rate
of oocytes collected from cattle on the low diet. A major problem with this type of
experiment is the limitation on the amount of material available from one animal at
slaughter. Further research from our laboratory suggests that the in-vitro blastocyst
yield of oocytes collected over several weeks using tram-vaginal ovum aspiration,
could be enhanced by restricting the dietary intake of heifers (67; Table 1). This
suggests that nutrition may affect reproduction at the level of the oocyte before
ovulation.

Table 1.Effect of diet quantity on mean (f SEM) number of follicles, and blastocyst
formation rate from oocytes’collected transvaginally and cultured in-vitro.
Heifers were offered a low (lkg/day of concentrates plus 3kg of hay) or high
(7kg/day of concentrates plus ad libitum hay) daily dietary allowance.
From Nolan et al. (67)
Dietary treatment Low High
Number of heifers 16 16
Total number of collections 72 72
No. follicles aspirated per collection 6.4 f 0.4’ 7.5 f 0.4b
No. oocytes recovered per collection 2.2 f 0.2 2.3 f 0.2
% oocytes cleaved (number) 73.0 (100/137)’ 61.8 (97/157)b
% blastocysts per oocyte cultured (number) 24.1 (331137)’ 12.7 (20/157)’
Within rows a, b P < 0.05; c, d P < 0.01.

Kendrick et al. (39) examined the effects of energy balance and postpartum
interval on the quality of oocytes in lactating dairy cows. Cows fed high-energy diets
had smaller follicles than cows offered low-energy diets, but cows o&red high-energy
diets produced more good quality oocytes. These results suggest that oocyte quality
is influenced by dietary intake, and the degree of the effect can be influenced by
lactational and physiological state. This highlights the importance of avoiding severe
changes in diet in the pre-mating period.

NUTRITION AND EMBRYO QUALITY

Short-term restriction in dietary intake has been shown to increase subsequent

pregnancy rates in cattle (21). Mantovani et al. (57) reported that the yield of
transferable embryos after superovulation in beef heifers was significantly reduced
when heifers had ad-libitw access to concentrates compared with restricted levels. In
addition concentrate type and quantity were shown to affect the subsequent yield of
transferable embryos after superovulation in beef heifers (96; Table 2).

Heifers on a restricted diet, where the predominant concentrate supplement was

in the form of citrus and beet pulp, produced more transferable embryos compared
 Theriogenology

with those where barley was the predominant concentrate or with those on ad-lib&urn
quantities of concentrates. Severe restriction in dietary intake (approximately 66% of
maintenance energy requirement) was shown to have beneficial effects on embryo
development rates when heifers were superovulated and embryos were collected 7
days after breeding and then cultured in vitro for 24 hours (67; Table 3). Nutrient
restriction resulted in an increase in the number of blastocysts after culturing embryos
for 24 h and an increase in the total cell number per blastocyst.

Table 2. Effect of concentrate intake (3 kg vs ad-libitum) and type (barley vs pulp)

on embryo yield (Mean rt SEM) after superovulation in heifers. From
Yaakub et al. (96)

Concentrate intake 3 ad-libitum Ha Citrus/beet pulo

No. heifers 39 37 38 38
No. corpora lutea 15.5*1.6a 12.3kI.4b 13.4+1.5 14.4kl.5
No. Ova/embryos 9.550.9c 6.5kO.9d 7.WO.9 8.UO.9
No.Grade 1 / 2 embryos 2.7M.5e 1.W0.3f 1.3*0.3a 2.4+0.5b
No. Grade 3 embryos 2.UO.3 1.8kOo.3 1.5AO.2 2.3kO.3

a b (P < 0.06); c d (P < 0.05); e f (P < 0.001).

In other studies, ewes were used as experimental models to determine the

effect of nutrition on embryo quality, and superovulation was used to increase the yield
of embryos. A lower percentage of good quality embryos was recovered on Day 4
from ewes on both a low diet (0.5 times the maintenance energy requirements) and an
ad libitum diet, compared with animals on a control diet (1.5 M; 49). However, while
the embryos from the ad-libitum group were delayed in their development compared
with those from ewes on the low diet; oocytes from the low dietary intake group had a
lower fertilization rate. Overall, due to a poorer response to the superovulatory
treatment in terms of the number of animals showing estrus and lower ovulation rate,
the embryo yield was lower in the ad libitum group when compared with the control
and the low group (49). It is possible that similar effects of high dietary intake and
metabolic load are affecting the quality of embryos produced in high yielding dairy
cows. Preliminary evidence in dairy cows suggests that embryo quality as recorded by
total cell number after recovery on Day 7, is reduced in cows offered large quantities
of food in early lactation (Snijders, unpublished).

The effect of extremes of dietary intake on embryo development is evident but

the point at which this change occurs is still unknown. Glucose intbsion also has been
reported to reduce pregnancy rates in ewes (83). Direct infusion of glucose to ewes
can increase ovulation rates (19), but embryo quality can be reduced dramatically (28,
99). It is known that high glucose concentrations are deleterious to embryo
development in-vitro (28). While the reason for such an effect of glucose on embryo
development is not clear, it may be due to unusually high plasma gh~cose
concentrations interfering with cell signalling through non-enzymatic protein glycation
during pre-ovulatory follicle growth, oocyte development or early embryo
development. There are also suggestions that hyperglycaemia in ewes is associated
with embryopathy (5 8).
Theriogenology 1331

Table 3. The effect of low (40 MJ ME per day) or high ( 120 MJ ME per day) dietary
intake on mean (ASEM) embryo yield on Day 7 in superovulated heifers, and
development capacity in-vitro. From Nolan et al. (67)
Diet Low High
No. heifers 14 13
No. of corpora tutea 16.3 f 3.0 14.4 f 1.9
No. of ova / embryos 11.4 f 2.4 10.4 f 1.3
No. of grade 1+2 embryos 4.5 * 1.3 3.5 f 1.1

Embrvo culture for 24 hours

Blastocyst % (number) 73 (78/107)a 41 (44/1o6)b
Blastocyst cell number 98.3 f 3.Oc 75.4 f 2.3d
Withinrows a,bP<O.Ol; c,dP<O.OOl.

Blastocyst formation is well recognised as a key developmental process in the

growth of an embryo. The blastocoel cavity forms as a consequence of fluid transport
across the trophectoderm. This process is partially facilitated by NaK-ATPase;
messenger RNA for this enzyme has been identified in Day 7 bovine embryos (18).
Dietary intake and diet type can alter the expression of transcripts of genes involved in
early embryo development, such aa Nan< ATPase and Cu/Zn SOD (94). Abecia et al.
(1) reported a decrease in the in vitro secretion of interferon tau in Day-15 embryos
from undernourished ewes. An increase in the in-vitro secretion of PGFz- by
endometrial tissue was evident in the same animals.

It is important to differentiate between optimum conditions for follicle growth,

(both in terms of number of follicles and paracrine environment), and optimum
conditions for embryo survival. Nutritional conditions may not be similar for both.
The effect of increased glucose on increasing ovulation rate and decreasing embryo
development in sheep has been identified above (19, 28, 99). Preimplantation mouse
embryos exposed to hyperglycaemia also have delayed development (64). High dietary
intakes that would be likely to alter circulating glucose concentrations have resulted in
reduced embryo quality in cattle (57, 96). It is not clear why high concentrations of
glucose have such deleterious effects, but it may be due to interactions among insulin,
glucose and glucose transport proteins during early embryo development. Before
blastocyst formation, metabolic reliance of embryos is on lactate and pymvate rather
than glucose (45). It has recently been demonstrated that the first marked increase in
oxidation of glucose occurred between the 12- and 16-cell stage, but the incorporation
of glucose increased steadily up to 15-fold between the zygote and blastocyst stage
(40). Glucose transport was increased in mouse embryos in response to glucose
deprivation, and both IGF-I and insulin can stimulate glucose uptake (72). These
authors also suggested that GLUT1 is the possible transporter in the mouse blastocyst,
and that GLUT1 may be an insulin-stimulated recruitment. Moley et al. (63) suggest
that elevated concentrations of glucose may alter activity of the Kreb’s cycle, resulting
in retarded embryo development. These types of observation indicate the significance
that changes in the glucose-insulin-IGF-1 axis may have on early embryo development.
Thus, nutrient requirements for optimum follicle growth and embryo development may
be quite different. This highlights the importance of diet around the time of mating and
1332 Theriogenology

in particular the significance of extreme overfeeding or underfeeding either pre- or

post-mating, in regulating pregnancy rates.

NUTRITION OF THE EARLY EMBRYO l-N VITRO

The literature reported above summarized a range of experiments conducted

predominantly in live animals. By their nature such experimental models are variable
and control of conditions is difficult. For example, a change in the diet offered to an
animal will not only alter nutrient supply locally, but will alter a range of other
endocrine and physiological parameters, any of which could ultimately intluence
embryo development. To reduce some of these inherent difficulties of working with
live animal models, the effects of nutrition on oocyte and embryo development also
were studied using in-vitro development models. This allows control of experimental
conditions after introducing the oocyte or embryo into the culture conditions and
allows study of specific current conditions or indeed specific historic environments
before introduction to the standard culture conditions. Such studies are particularly
useful in studying early embryo development as part of a normal physiological process,
which requires the oocyte to develop in a definite follicular environment and
subsequently to be exposed to a complex milieu of oviduct and uterine fluid secretions.

Significant progress has been made in recent years in understanding conditions

required for embryo development in vitro. The role of nutrients such as energy
substrates and amino acids in controlling embryo development is complex and has been
the subject of several reviews (29, 30, 37, 90). It is becoming increasingly clear that
the starting point of embryo culture during in vitro culture, the zygote, has quite
different physiological needs to those of the blastocyst, the end-point of the pre-
implantation period. Changes in physiology as the embryo develops are reflected in
different nutrient requirements and energy metabolism. The early cleavage stage
embryo is characterized by relatively low levels of biosynthesis, low respiratory rates
and a limited capacity to use glucose as an energy source (32, 40). An increase in
biosynthetic rates along with increased respiration and ability of the embryo to use
glucose occurs a&r compaction. The ruminant embryo, before compaction, also
exhibits low levels of oxidative metabolism and oxygen consumption, whereas the later
stages exhibit both high levels of glycolysis and high oxygen consumption (90). It is
therefore rather paradoxical that most of the efforts to optimize culture media have
focused on the development of a single medium to support all stages of development.
In fact it is only quite recently that work has been carried out using sequential culture
media (30) or a continuous perfusion culture system (90) to take account of the
changing requirements of the developing embryo.

Carbohydrate Effects on Embryo Development

Energy substrates are among the most important ingredients of any culture
medium. A substantial body of evidence highlights the changing energy substrate
requirements during early embryo development with important differences among
species (9, 29, 46, 81). Several studies examine the carbohydrate use and requirements
for bovine and ovine embryo development. As with most other mammalian species,
use generahy increases as development progresses, especially with the onset of
compaction. This is likely due to the high demand for energy placed by the Na+, K+-
Theriogenology

ATPase system required for blastocoel cavity formation. The ruminant embryo has a
limited capacity to utilize glucose before morula compaction occurs (82, 90). In fact,
up to this stage, plasma concentrations of gh~cose (5-6 mM) can be detrimental to
development (90). Instead, the early embryo uses pytuvate and lactate alone or in
combination with amino acids (10, 3 1, 46) to generate its required energy. Khurana
and Niemann (40) compared the metabolism of in-vitro and in-vivo derived bovine
embryos in parallel. Several distinct metabolic differences were identified including the
production of lactate and high rates of oxidation of energy substrates by in-vitro
matured oocytes. Similarly, in-vitro produced blastocysts produced twice as much
lactate as their in-vivo counterparts, highlighting the fact that all the requirements of
the developing embryo may not be met in vitro.

Amino Acids and Embryo Development

One of the most significant findings affecting embryo culture media formulation
in recent years was the report of beneficial effects after supplementation of groups of
amino acids (essential and nonessential groups). Amino acids are among the most
important regulators of pre-implantation development and therefore among the key
constituents of culture media. Inclusion of specific amino acids in embryo culture
media was shown to overcome the so-called “developmental blocks” observed in many
species It is clear that there are specific changes in nitrogen requirements of the
embryo. Three possible modes of action of amino acids have been proposed and these
include: regulators of energy metabolism; osmolytes and buffers of internal pH (30).

In a study ‘by Partridge and Leese (75) the depletion of 19 amino acids from
culture medium was measured during culture of bovine embryos, The rate of depletion
of individual amino acids changed with developmental stage, suggesting that the
bovine embryo changes its requirements for amino acids during development. Similar
findings were reported in the mouse (43). Steeves and Gardner (88) demonstrated that
the embryo has a switch ins its requirements for amino acids as it develops Corn the
zygote to the blastocyst. Development of the early cleavage stages were stimulated by
the nonessential amino acids and ghnamine, while development beyond Day 4 was
stimulated by a combination of the nonessential and essential amino acids and
glutamine. One potential disadvantage is that in culture, amino acids are metabolized
by the embryo and also spontaneously undergo breakdown to release ammonium into
the medium, the concentration of which increases with time (3 1). Ammonium is toxic
to embryos and therefore, in order for the amino acids to confer maximal benefit, the
culture medium must be replaced at regular intervals.

In summary, it is clear now that nutrition of the early embryo can have a
profound effect on its subsequent development. For example, until recently, studies of
the impact of nutrition on the growth of the conceptus concentrated on late pregnancy.
However, it is now evident that effects on fetal growth, the causes of which are likely
to be nutritional in origin, can be programmed very early in development. This
observation comes from in vitro embryo culture systems the composition of which can
influence subsequent fetal growth and gestation length (100). Thus, in vitro studies
such as those described above are essential to further our understanding of the factors
necessary for embryo development in viva.
1334 Theriogenology

CONCLUDING REMARKS

Although dietary intake can clearly influence reproductive i&ztion, the

relationship between nutrition and reproduction is complex. High dietary intake exerts
a negative effect on the developmental capacity of embryos. Experimental diets
offered below maintenance requirements resulted in improved embryo development
compared with animals on ad-libitum diets in several experiments from this laboratory.
The effects of nutrition are exerted very early in embryo development, possibly before
fertilization during the acquisition of developmental competence by the oocyte. This
negative effect of high dietary intake or metabolic load on fertility is a tindamental
challenge that needs to be addressed in particular in high production cows, where
fertility may be severely compromised. A key issue in improving reproductive
efficiency and embryo production may be to supply the nutritional needs of the animal
in a physiological way and avoid abnormal or unbalanced amounts of any one
component in the diet. Thus, nutritional balancing is critical for high-production
animals, in particular. The challenge remains to modifjl nutritional and management
strategies in such animal production systems to maintain the levels of production that
can now be achieved as a consequence of genetic selection and still maintain an
acceptable level of fertility.

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